Overexpression of aminoacyl-tRNA synthetases for efficient production of engineered proteins containing amino acid analogues

ABSTRACT

Methods for producing modified polypeptides containing amino acid analogues are disclosed. The invention further provides purified dihydrofolate reductase polypeptides, produced by the methods of the invention, in which the methionine residues have been replaced with homoallyglycine, homoproparglycine, norvaline, norleucine, cis-crotylglycine, trans-crotylglycine, 2-aminoheptanoic acid, 2-butynylglycine and allylglycine.

[0001] This application is based on and claims the priority of U.S. Ser.No. 60/207,627 filed May 26, 2000, the contents of which are herebyincorporated by reference in their entirety.

[0002] This invention was made with Government support under NSF GrantNos. NSF DMR-9996048 and US Army Research Grant DAAG55-98-1-0518. TheGovernment has certain rights in this invention.

[0003] Throughout this application various publications are referenced.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF INVENTION

[0004] The present invention relates to novel compositions and methods,for incorporating amino acid analogues into proteins in vivo, byoverexpression of aminoacyl-tRNA synthetases.

BACKGROUND OF INVENTION

[0005] Expanding the scope of biological polymerizations to includenon-natural monomers, is an area of growing interest, with importanttheoretical and practical consequences. An early and criticallyimportant example of such studies was the demonstration that “dideoxy”nucleotide monomers can serve as substrates for DNA polymerases.Advances in DNA sequencing (F. Sanger, S. Nicklen, A. R. Coulson, Proc.Natl. Acad. Sci. USA 1977, 74, 5463-5467), DNA base pairing models (M.J. Lutz, S. A. Benner, S. Hein, G. Breipohl, E. Uhlmann, J. Am. Chem.Soc. 1997, 119, 3177-3178; J. C. Morales, E. T. Kool, Nature Struct.Biol. 1998, 5, 950-954), materials synthesis (W. H. Park, R. W. Lenz, S.Goodwin, Macromolecules 1998, 31, 1480-1486; Y. Doi, S. Kitamura, H.Abe, Macromolecules 1995, 28, 4822-4828), and cell surface engineering(K. J. Yarema, L. K. Mahal, R. E. Bruehl, E. C. Rodriguez, C. R.Bertozzi, J. Biol. Chem. 1998, 273, 31168-31179; L. K. Mahal, K. J.Yarema, C. R. Bertozzi, Science 1997, 276, 1125-1128; Saxon, E. andBertozzi, C. R. Science 2000, 287, 2007-2010) have resulted from therecognition of non-natural monomers by the enzymes that control thesepolymerizations.

[0006] Recent investigations have shown the incorporation of modified orcompletely “synthetic” bases into nucleic acids (Matray, T. J.; Kool, E.T. Nature 1999, 399, 704; Kool, E. T. Biopolymers 1998, 48, 3; Morales,J. C. ; Kool, E. T. Nature Struct. Biol. 1998, 5, 950; Guckian, K. M. ;Kool, E. T. ; Angew. Chem. Int. Ed. Eng. 1998, 36, 2825; Liu, D. Y. ;Moran, S.; Kool, E. T. Chem. Biol. 1997, 4, 919; Moran, S.; Ren, R. X.F.; Kool, E. T. Proc. Natl. Acad. Sci. USA 1997, 94, 10506; Moran, S. etal. J. Am. Chem. Soc. 1997, 119, 2056; Benner, S. A. et al. Pure Appl.Chem. 1998, 70, 263; Lutz, M. J.; Horlacher J.; Benner, S. A. Bioorg.Med. Chem. Lett. 1998, 8, 1149; Lutz, M. J. ; Held, H. A. ; Hottiger,M.; Hubscher, U.; Benner, S. A. Nuc. Acids Res. 1996, 24, 1308;Horlacher, J. et al. Proc. Natl. Acad. Sci. USA 1995, 92, 6329; Switzer,C. Y. ; Moroney, S. E. ; Benner, S. A. Biochemistry 1993, 32, 10489;Lutz, M. J.; Horlacher, J.; Benner, S. A. Bioorg. Med. Chem. Lett. 1998,8, 499; Switzer, C.; Moroney, S. E. ; Benner, S. A. J. Am. Chem. Soc.1989, 111, 8322; Piccirilli, J. A. ; Krauch, T.; Moroney, S. E. ;Benner, S. A. Nature 1990, 343, 33), while materials researchers haveexploited the broad substrate range of the poly(β-hydroxyalkanoate)(PHA) synthases to prepare novel poly(β-hydroxyalkanoate)s (PHAs) withunusual physical properties (Kim, Y. B.; Rhee, Y. H.; Lenz, R. W. Polym.J. 1997, 29, 894; Hazer, B.; Lenz, R. W.; Fuller, R. C. Polymer 1996,37, 5951; Lenz, R. W. ; Kim, Y. B. ; Fuller, R. C. FEMS Microbiol. Rev.1992, 103, 207; Park, W. H.; Lenz, R. W.; Goodwin, S. Macromolecules1998, 31, 1480; Ballistreri, A. et al. Macromolecules 1995, 28, 3664;Doi, Y.; Kitamura, S.; Abe, H. Macromolecules 1995, 28, 4822).

[0007] Novel polymeric materials with unusual physical and/or chemicalproperties are also useful in polymer chemistry. The last severaldecades have shown many advances in synthetic polymer chemistry thatprovide the polymer chemist with increasing control over the structureof macromolecules (Szwarc, M. Nature 1956, 178, 1168-1169 Szwarc, M.Nature 1956, 178, 1168-1169; Faust, R.; Kennedy, J. P. Polym. Bull.1986, 15, 317-323; Schrock, R. R. Acc. Chem. Res. 1990, 23, 158-165;Corradini, P. Macromol Symp. 1995, 89, 1-11; Brintzinger, H. H.;Fischer, D.; Mulhaupt, R.; Rieger, B.; Waymouth, R. M. Angew. Chem. Int.Ed. Engl. 1995, 34, 1143-1170; Dias, E. L.; SonBinh, T. N.; Grubbs, R.H. J. Am. Chem. Soc. 1997, 119, 3887-3897; Chiefari, J. et al.Macromolecules 1998, 31, 5559-5562). However, none of these methods haveprovided the level of control that is the basis of the exquisitecatalytic, informational, and signal transduction capabilities ofproteins and nucleic acids (Ibba, M.; Soll, D. Science 1999, 286,1893-1897). There remains a need for control over protein synthesis todesign and produce artificial proteins having advantageous properties.

[0008] For this reason, the design and synthesis of artificial proteinsthat exhibit novel and potentially useful structural properties havebeen investigated. Harnessing the molecular weight and sequence controlprovided by in vivo synthesis would permit control of folding,functional group placement, and self-assembly at the angstrom lengthscale. Proteins that have been produced by in vivo methods exhibitpredictable chain-folded lamellar architectures (Krejchi, M. T.; Atkins,E. D. T.; Waddon, A. J.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A.Science 1994, 265, 1427-1432; Parkhe, A. D.; Fournier, M. J. ; Mason, T.L.; Tirrell, D. A. Macromolecules 1993, 26(24), 6691-6693; McGrath, K.P.; Fournier, M. J. ; Mason, T. L.; Tirrell, D. A. J. Am. Chem. Soc.1992, 114, 727-733; Creel, H. S.; Fournier, M. J.; Mason, T. L.;Tirrell, D. A. Macromolecules 1991, 24, 1213-1214), unique smecticliquid-crystalline structures with precise layer spacings (Yu, S. M.;Conticello, V.; Zhang, G.; Kayser, C.; Fournier, M. J.; Mason, T. L.;Tirrell, D. A. Nature 1997, 389, 187-190), and controlled reversiblegelation (Petka, W. A.; Hardin, J. L.; McGrath, K. P.; Wirtz, D.;Tirrell, D. A. Science 1998, 281, 389-392). The demonstrated ability ofthese protein polymers to form unique macromolecular architectures willbe of importance for engineering materials with interestingliquid-crystalline, crystalline, surface, electronic, and opticalproperties.

[0009] Novel chemical and physical properties that can be engineeredinto protein polymers may be expanded by the precise placement of aminoacid analogues. Efforts to incorporate novel amino acids into proteinsin vivo have relied on the ability of the translational apparatus torecognize amino acid analogues that differ in structure andfunctionality from the natural amino acids. The in vivo incorporation ofamino acid analogues into proteins is controlled most stringently by theaminoacyl-tRNA synthetases (AARS), the class of enzymes that safeguardsthe fidelity of amino acid incorporation into proteins (FIG. 1). The DNAmessage is translated into an amino acid sequence via the pairing of thecodon of the messenger RNA (mRNA) with the complementary anticodon ofthe aminoacyl-tRNA. Aminoacyl-tRNA synthetases control the fidelity ofamino acid attachment to the tRNA. The discriminatory power of theaminoacyl-tRNA synthetase places severe limits on the set of amino acidstructures that can be exploited in the engineering of natural andartificial proteins in vivo.

[0010] Several strategies for circumventing the specificity of thesynthetases have been explored. Introduction of amino acid analogues canbe achieved relatively simply via solid-phase peptide synthesis(Merrifield, R. B. Pure & Appl. Chem. 1978, 50, 643-653). While thismethod circumvents all biosynthetic machinery, the multistep procedureis limited to synthesis of peptides less than or equal to approximately50 amino acids in length, and is therefore not suitable for producingprotein materials of longer amino acid sequences.

[0011] Chemical aminoacylation methods, introduced by Hecht andcoworkers (Hecht, S. M. Acc. Chem. Res. 1992, 25, 545; Heckler, T. G.;Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M. Biochemistry 1988, 27,7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.; Kitano, S. J. Biol. Chem.1978, 253, 4517) and exploited by Schultz, Chamberlin, Dougherty andothers (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew. Chem. Int. Ed.Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J.Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.; Anthony-Cahill, S. J.;Griffith, M. C.; Schultz, P. G. Science 1989, 244, 182; Bain, J. D.;Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J. Am. Chem. Soc. 1989, 111,8013; Bain, J. D. et al. Nature 1992, 356, 537; Gallivan, J. P.; Lester,H. A.; Dougherty, D. A. Chem. Biol. 1997, 4, 740; Turcatti, et al. J.Biol. Chem. 1996, 271, 19991; Nowak, M. W. et al. Science, 1995, 268,439; Saks, M. E. et al. J. Biol. Chem. 1996, 271, 23169; Hohsaka, T. etal. J. Am. Chem. Soc. 1999, 121, 34), avoid the synthetases altogether,but provide low protein yields.

[0012] Alteration of the synthetase activities of the cell is alsopossible, either through mutagenesis or through introduction ofheterologous synthetases (Ibba, M.; Hennecke, H. FEBS Lett. 1995, 364,272; Liu, D. R.; Maghery, T. J.; Pastmak, M.; Schultz, P. G. Proc. Natl.Acad. Sci. USA, 1997, 94, 10092; Furter, R. Protein Sci. 1998, 7, 419;Ohno, S. et al., J. Biochem. 1998, 124, 1065; Liu, D. R.; Schultz, P. G.Proc. Natl. Acad Sci. 1999, 96, 4780; Wang, L.; Magliery, T. J.; Liu, D.R.; Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 5010-5011; Pastrnak, M.;Magliery, T. J.; Schultz, P. G. Helv. Chim. Acta 2000, 83, 2277-2286).

[0013] In some instances, the ability of the wild-type synthetases toaccept amino acid analogues has been exploited. For example, wild-typesynthetases have been shown to activate and charge substrates other thanthe canonical, proteinogenic amino acids (Cowie, D. B.; Cohen, G. N.Biochim. Biophys. Acta. 1957, 26, 252; Richmond, M. H. Bacteriol Rev.1962, 26, 398; Horton, G.; Boime, I. Methods Enzymol. 1983, 96, 777;Wilson, M. J.; Hatfield, D. L. Biochim. Biophys. Acta 1984, 781, 205).This approach offers important advantages with respect to syntheticefficiency, in that neither chemical acylation of tRNA nor cell-freetranslation is required. The simplicity of the in vivo approach, itsrelatively high synthetic efficiency, and its capacity for multisitesubstitution, make it the method of choice for production of proteinmaterials whenever possible.

[0014] The capacity of the wild-type translational apparatus has beenpreviously demonstrated to utilize amino acid analogues bearingfluorinated (Richmond, M. H. J. Mol. BioL 1963, 6, 284; Fenster, E. D.;Anker, H. S. Biochemistry 1969, 8, 268; Yoshikawa, E.; Fournier, M. J.;Mason, T. L.; Tirrell, D. A. Macromolecules 1994, 27, 5471), unsaturated(Van Hest, J. C. M.; Tirrell, D. A. FEBS Lett. 1998, 428, 68; Deming, T.J.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. J. Macromol. Sci.-Pure AppL. Chem. 1997, A34, 2134), electroactive (Kothakota, S.; Mason,T. L.; Tirrell, D. A.; Fournier, M. J. J. Am. Chem. Soc. 1995, 117,536), and other useful side chain functions. The chemistries of theabove functional groups are distinct from the chemistries of the amine,hydroxyl, thiol, and carboxylic acid functional groups characteristic ofproteins; this makes their incorporation particularly attractive fortargeted chemical modification of proteins.

[0015] For example, alkene functionality introduced into artificialproteins via dehydroproline can be quantitatively modified viabromination and hydroxylation (Deming, T. J.; Fournier, M. J.; Mason, T.L.; Tirrell, D. A. J. Macromol. Sci. Pure Appl. Chem. 1997, A34,2143-2150). Alkene functionality, introduced by incorporation of otheramino acid analogues, should be useful for chemical modification ofproteins by olefin metathesis (Clark, T. D.; Kobayashi, K.; Ghadiri, M.R. Chem. Eur. J. 1999, 5, 782-792; Blackwell, H. E.; Grubbs, R. H.Angew. Chem. Int. Ed. Engl. 1998, 37, 3281-3284), palladium-catalyzedcoupling (Amatore, C.; Jutand, A. J. Organomet. Chem. 1999, 576,255-277; Tsuji, J. Palladium Reagents and Catalysts. Innovations inOrganic Synthesis; John Wiley and Sons: New York, 1995; Schoenberg, A.;Heck, R. F. J. Org. Chem. 1974, 39, 3327-3331), and other chemistries(Trost, B. M.; Fleming, I., Eds. Comprehensive Organic Synthesis;Pergamon Press: Oxford, 1991). The incorporation of fluorinatedfunctional groups into proteins has imparted to protein films the lowsurface energy characteristic of fluoropolymers; contact angles ofhexadecane on fluorinated protein polymers (70°) are much higher thanthose on unfluorinated controls (17°) (Yoshikawa, E.; Fournier, M. J.;Mason, T. L.; Tirrell, D. A. Macromolecules 1994, 27, 5471-5475).

[0016] Methionine (1) (FIG. 1) is a possible target for substitution byamino acid analogues, with its hydrophobicity and polarizability, makeit an important amino acid for regulating protein structure andprotein-protein recognition processes (T. Yuan, A. M. Weljie, H. J.Vogel, Biochemistry 1998, 37, 3187-3195; H. L. Schenck, G. P. Dado, S.H. Gellman, J. Am. Chem. Soc. 1996, 118, 12487-12494; Maier, K. L.;Lenz, A. G., Beck-Speier, I.; Costabel, U. Methods Enzymol. 1995, 251,455-461). Replacement of methionine by its analogues may thereforepermit purposeful manipulation of these properties.

[0017] Several analogues of methionine (1), specificallyselenomethionine, telluromethionine, norleucine, trifluoromethionine andethionine (Hendrickson, W. A.; Horton, J. R.; Lemaster, D. M. EMBO J.1990, 9, 1665; Boles, J. O. et al Nature Struct. Biol. 1994, 1, 283;Cowie, D. B.; Cohen, G. N.; Bolton, E. T.; de Robichon-Szulmajster, H.Biochim. Biophys. Acta 1959, 34, 39; Duewel, H.; Daub, E.; Robinson, R.;Honek, J. F. Biochemistry 1997, 36, 3404; Budisa, N.; Steipe, B.;Demange, P.; Eckerskom, C.; Kellerman, J.; Huber, R. Eur. J. Biochem.1995, 230, 788), have been shown to exhibit translational activity inbacterial hosts. Incorporation of selenomethionine in place ofmethionine has long been known to facilitate protein structuredetermination by x-ray crystallography (Wei, Y.; Hendrickson, W. A.;Crouch, R. J.; Satow, Y. Science 1990, 249, 1398-1405).

[0018] However, only a limited number of amino acid analogues have beenshown to conclusively exhibit translational activity in vivo, and therange of chemical functionality accessible via this route remainsmodest. These circumstances dictate a need for a systematic search fornew amino acid analogues and strategies that will allow the engineeringof proteins with novel chemical and physical properties.

SUMMARY OF INVENTION

[0019] The present invention seeks to overcome these and otherdisadvantages in the prior art by providing a novel method forincorporating amino acid analogues into polypeptides of interest in vivoby expanding the scope of amino acid analogues that are incorporated andincreasing protein yields. Preferably, the production of modifiedpolypeptides can be in a host-vector system in which a natural aminoacid in the wild-type polypeptide is replaced with a selected amino acidanalogue by overexpressing an aminoacyl-tRNA synthetase corresponding tothe natural amino acid so replaced.

[0020] In addition, the present invention provides novel host-vectorsystems. The host-vector system produces an aminoacyl-tRNA synthetase inan amount in excess of the level of a naturally occurring aminoacyl-tRNAsynthetase. The system also produces a polypeptide of interest in anamount in excess of the level produced by a naturally occurring geneencoding the polypeptide of interest.

[0021] Nucleic acids encoding the expression vectors, hosts, and methodsof integrating a desired amino acid analogue into target polypeptidesare also provided.

[0022] The invention further provides purified dihydrofolate reductasepolypeptides, produced by the methods of the invention, in which themethionine residues have been replaced with homopropargylglycine(2-amino-hexynoic acid), homoallylglycine (2-amino-hexenoic acid),cis-crotylglycine (cis-2-amino-4-hexenoic acid), trans-crotylglycine(trans-2-amino-4-hexenoic acid), norleucine, 6,6,6-trifluoro-2-aminohexanoic acid, 2 -amino-heptanoic acid, norvaline, o-allylserine,2-butynylglycine, allylglycine or propargylglycine. The formation of themodified polypeptides demonstrate the ease and efficiency of the methodsof the invention for incorporating amino acid analogues such as,methionine analogues, into proteins such as, dihydrofolate reductase.

[0023] Using the methods of the invention, it is possible to produceentirely new polypeptides containing amino acid analogues having unusualproperties.

BRIEF DESCRIPTION OF THE FIGURES

[0024]FIG. 1 depicts a schematic diagram of in vivo protein synthesis.

[0025]FIG. 2 depicts a set of methionine analogues (2-13), as describedin Example I, infra. FIG. 3 illustrates the SDS-PAGE analysis of mDHFRsynthesis by E. coli strain CAG18491/pREP4/pQE15, as described inExample I, infra. Cultures were supplemented with methionine or with oneof the analogues 2-9, as indicated. Each lane is identified in terms ofthe time of analysis subsequent to addition of the inducer IPTG. mDHFRis visualized by staining with Coomassie Brilliant Blue. The targetprotein can be detected only in cultures supplemented with methionine orwith analogues 2, 3, or 9, respectively.

[0026]FIG. 4 shows the determination of the occupancy of the initiatorsite in: a). mDHFR, b). mDHFR-E (alkene) and c). mDHFR-Y(alkyne), asdescribed in Example I, infra. Chromatograms are shown for analysis ofthe N-terminal residue in each of the three proteins, as determined viaEdman degradation. The signals corresponding to methionine, 2 and 3elute at 12.3, 14.3 and 11.0 min, respectively. The strong signal at13.8 min is due to piperidylphenylthiourea, a by-product of theanalysis. Signals assigned to 2 and 3 were verified by analysis ofauthentic samples of the analogues.

[0027]FIG. 5 depicts the activation of methionine and methionineanalogues by MetRS (Methionyl tRNA synthetase), as described in ExampleI, infra. The amount of PP₁ exchanged in 20 minutes is shown formethionine (1) and for methionine analogues 2-13. The background (14) isgiven for a reaction mixture lacking both enzyme and amino acid.

[0028]FIG. 6 illustrates the electron density maps (colored surfaces)and negative isopotential surfaces (meshes) for methionine (a) and foranalogues 2, 3 and 5 (b-d, respectively), as described in Example I,infra. The electron density maps indicate electron-rich (red) andelectron-poor (blue) regions of each molecule. For simplicity, the aminoacid form is shown; this avoids representation of the highly extendedisopotential surface of the carboxylate anion of the zwitterion andfacilitates comparison of side-chain electronic structure.

[0029]FIG. 7 shows the SDS-PAGE analysis of mDHFR synthesis by E. colistrains B834(DE3)/pQE15/pREP4 (designated pQE15) andB834(DE3)/pQE15-MRS/pREP4 (designated pQEI5-MRS), as described inExample II, infra. Cultures (M9+19AA) were supplemented with nothing(−Met), methionine (Met) or trans-crotylglycine (60 mg/L) (Tcg), asindicated.

[0030]FIG. 8 depicts the SDS-PAGE analysis of DHFR synthesis by E. colistrains CAG18491/pQE15/pREP4 and CAG18491/pQE15-MRS/pREP4, as describedin Example II, infra. Cultures (M9+19AA) were supplemented with nothing(−Met), methionine (+Met), or 2-butynylglycine (60 mg/L) (+2bg), asindicated.

[0031]FIG. 9 shows the SDS-PAGE analysis of mDHFR synthesis by E. colistrains CAG18491/pQE15/pREP4 (the left panel in each pair, pQEl5) andCAG18491/pQE15-MRS/pREP4 (the right panel in each pair, pQE15-MRS), asdescribed in Example II, infra. Cultures (M9+19AA) were supplementedwith the analogues 4, 6, 7, 8, 10, 12, and 13 at 500 mg/l, as indicated.Negative (−Met) and positive (+Met) controls of CAG18491/pQE15-MRS/pREP4cultures are also shown for comparison

[0032]FIG. 10 is a table detailing kinetic parameters for methionineanalogues in the ATP-PP₁ exchange reaction and analogue's ability tosupport protein biosynthesis in cultures of a conventional bacterialhost supplemented with the analogues, as described in Example II, infra.

[0033]FIG. 11 illustrates the activation rates of methionine by wholecell lysates, as described in Example II, infra. Maximum ATP-PPIexchange velocities, measured at a saturating concentration ofmethionine (750 μM), are shown for whole cell lysates ofB834(DE3)/pQE15/pREP4 (solid) and of B834(DE3)/pQE15-MRS/pREP4(striped). Rates were measured for (a) cell lysates obtained fromcultures prior to protein expression, (b) cell lysates obtained fromcultures supplemented with methionine during protein expression, and (c)cell lysates obtained from cultures supplemented with transcrotylglycineduring protein expression.

[0034]FIG. 12 shows the Proton NMR spectra (599.69 MHz) of (a) mDHFR,(b) Tcg, (c and d) mDHFR-Tcg, as described in Example II, infra. Sampleswere dissolved at concentrations of approximately 10 mg/ml in D₂Ocontaining 2% d-formic-d-acid and spectra were collected at 25° C.overnight.

[0035]FIG. 13 depicts the N-terminal sequencing results indicatingoccupancy of the initiator site in mDHFR-Tcg, as described in ExampleII, infra. Chromatograms are shown for (a) the N-terminal residue ofmDHFR, (b) Tcg control, and (c) the N-terminal residue of mDHFR-Tcg, asdetermined via Edman degradation.

[0036]FIG. 14 depicts the N-terminal sequencing results indicatingoccupancy of the initiator site in mDHFR-bg, as described in Example II,infra. Chromatograms are shown for (a) the N-terminal residue of mDHFR,(b) 2bg control, and (c) the N-terminal residue of mDHFR-2bg, asdetermined via Edman degradation.

[0037]FIG. 15 is a table of the kinetic parameters for methionineanalogues in the ATP-PP, exchange reaction and protein yields forbacterial cultures supplemented with the analogues, as described inExample JII, infra.

[0038]FIG. 16 shows the comparison of the kinetic parameters formethionine analogues in the ATP-PP₁ exchange reaction and relativeprotein yields from conventional bacterial host cultures supplementedwith the analogues, as described in Example III, infra.

[0039]FIG. 17 depicts the Western blot analysis of protein synthesis bybacterial expression hosts CAG18491/pQE15/pREP4 (pQE15) andCAG18491/pQE15-MRS/pREP4 (MRS). Bacterial cultures were supplementedwith methionine, 2, 3 or 9, as described in Example III, infra.

[0040]FIG. 18 illustrates the activation (a) and aminoacylation (b)steps of amino acid attachment to tRNA, as described in Example III,infra.

[0041]FIG. 19 depicts the sequence of pQE15-MRS (SEQ ID NO.: 1).

[0042]FIG. 20 depicts the sequence of pQEI5-W305F (SEQ ID NO.: 2)

DETAILED DESCRIPTION OF THE INVENTION

[0043] As used in this application, the following words or phrases havethe meanings specified.

[0044] Definitions

[0045] As used herein, a polypeptide refers to a peptide or proteinhaving natural amino acids.

[0046] As used herein, modified polypeptides are polypeptides havingamino acid analogues incorporated into their amino acid sequence.

[0047] As used herein, a “natural amino acid” is one of the 20 naturallyoccuring amino acids, namely glycine, alanine, valine, leucine,isoleucine, serine, threonine, aspartic acid, glutamic acid, asparagine,glutamine, lysine, arginine, cysteine, methionine, phenylalanine,tyrosine, tryptophan, histidine and proline.

[0048] As used herein, the term “amino acid analogue” refers to acompound that has a structure analogue to a natural amino acid butmimics the structure and/or reactivity of a natural amino acid. Thisincludes all amino acids but the natural 20 amino acids are referred toas amino acid analogues even if they are naturally present (e.g.hydroxyproline).

[0049] As used herein, the term “peptide” refers to a class of compoundscomposed of amino acids chemically bound together with amide linkages(CONH). Peptide as used herein includes oligomers of amino acids andsmall and large peptides, including polypeptides.

[0050] As used herein, “polypeptides” embrace all peptides and thosepolypeptides generally defined as proteins and also those that areglycosylated, e.g. glycoproteins.

METHODS OF THE INVENTION

[0051] The present invention is based on the discovery thatincorporation of amino acid analogues into polypeptides can be improvedin cells that overexpress aminoacyl-tRNA synthetases that recognizeamino acid analogues as substrates. “Improvement” is defined as eitherincreasing the scope of amino acid analogues (i.e. kinds of amino acidanalogues) that are incorporated or by increasing the yield of themodified polypeptide. Overexpression of the aminoacyl-tRNA synthetaseincreases the level of aminoacyl-tRNA synthetase activity in the cell.The increased activity leads to an increased rate of incorporation ofamino acid analogues into the growing peptide, thus the increased rateof synthesis of the polypeptides, thereby increasing the quantity ofpolypeptides containing amino acid analogues, i.e. modifiedpolypeptides, produced.

[0052] In general, the methods of the invention comprises introducinginto a host cell, a vector having nucleic acids encoding anaminoacyl-tRNA synthetase, and nucleic acids encoding a polypeptide ofinterest to produce a host-vector system. The nucleic acids, encodingthe aminoacyl-tRNA synthetase, and the nucleic acids encoding thepolypeptide of interest, may be located in the same or differentvectors. The vectors include expression control elements which directthe production of the aminoacyl-tRNA synthetase, and the polypeptide ofinterest. The expression control elements (i.e. regulatory sequences)can include inducible promotors, constitutive promoters, secretionsignals, enhancers, transcription terminators, and other transcriptionalregulatory elements.

[0053] In the host-vector system, the production of an aminoacyl-tRNAsynthetase can be controlled by a vector which comprises expressioncontrol elements that direct the production of the aminoacyl-tRNAsynthetase. Preferably, the production of aminoacyl-tRNA synthetase isin an amount in excess of the level of naturally occurringaminoacyl-tRNA synthetase, such that the activity of the aminoacyl-tRNAsynthetase is greater than naturally occuring levels.

[0054] In the host-vector system, the production of a polypeptide ofinterest can be controlled by a vector which comprises expressioncontrol elements for producing the polypeptide of interest. Preferably,the polypeptide of interest so produced is in an amount in excess of thelevel produced by a naturally occurring gene encoding the polypeptide ofinterest.

[0055] The host-vector system can be constitutively overexpressing theaminoacyl-tRNA synthetase and induced to overexpress the polypeptide ofinterest by contacting the host-vector system with an inducer, such asisopropyl-β-D-thiogalactopyranoside (IPTG). The host-vector system canalso be induced to overexpress the aminoacyl-tRNA synthetase and/or theprotein of interest by contacting the host-vector system with aninducer, such as IPTG. Other inducers include stimulation by an externalstimulation such as heat shock.

[0056] Using the methods of the invention, any natural amino acid can beselected for replacement by an amino acid analogue in the polypeptide ofinterest. An amino acid analogue is preferably an analogue of thenatural amino acid to be replaced. To replace a selected natural aminoacid with an amino acid analogue in a polypeptide of interest, anappropriate corresponding aminoacyl-tRNA synthetase must be selected.For example, if an amino acid analogue will replace a methionineresidue, then preferably a methionyl tRNA synthetase is selected.

[0057] The host-vector system is grown in media lacking the naturalamino acid and supplemented with an amino acid analogue, therebyproducing a modified polypeptide that has incorporated at least oneamino acid analogue. This method is superior to existing methods as itimproves the efficiency of incorporation of amino acid analogues intopolypeptides of interest and increases the quantity of modifiedpolypeptides so produced.

[0058] In an embodiment of the invention, where the host-vector systemis an auxotrophic system, the host-vector system is initially grown inmedia which includes all essential amino acids, induced to express thepolypeptide of interest, and subsequently after induction, is grown inmedia lacking the natural amino acid and supplemented with an amino acidanalogue, thereby producing a modified polypeptide that has incorporatedat least one amino acid analogue.

[0059] For example, the method of the invention can be practiced by: (1)growing the host-vector system under suitable conditions having thenatural amino acid and under conditions such that the host-vector systemoverexpresses the aminoacyl-tRNA synthetse; (2) collecting and washingcells to remove presence of the natural amino acid; (3) resuspending thecells in media medium which lacks the natural amino acid and has anamino acid analogue; (4) inducing the expression of the polypeptide ofinterest; (5) growing the cells in a medium which lacks the naturalamino acid and has an amino acid analogue under conditions such that thehost-vector system overexpresses the aminoacyl-tRNA synthetase and thepolypeptide molecule of interest; and (6) isolating the modifiedpolypeptide of interest.

[0060] In an embodiment of the invention, the polypeptide of interest isdihydrofolate reductase, the natural amino acid is methionine, theaminoacyl-tRNA synthetase is methionyl tRNA synthetase, and the aminoacid analogues of methionine are 6,6,6-trifluoromethionine,homoallyglycine, homoproparglycine, norvaline, norleucine,cis-crotylglycine, trans-crotylglycine, 2-aminoheptanoic acid,2-butynylglycine, allylglycine, azidoalanine and azidohomoalanine.

[0061] Polypeptides of Interest

[0062] In accordance with the invention, the polypeptides may be fromany source whether natural, synthetic, semi-synthetic, or recombinant.These include hormones, enzymes and protein fibers. Of these proteins,well-known examples are insulin, interferons, growth hormones, serumalbumin and epidermal growth factor.

[0063] The polypeptides of interest can be those which wild-type cellscannot naturally produce. In view of the diversity of the modifiedpolypeptides that can be produced using the methods of the invention, itis preferable that the polypeptide of interest be different from thoseproduced by wild type cells.

[0064] Natural Amino Acids

[0065] Natural amino acids are amino acid residues that will be replacedin a polypeptide of interest by a desired amino acid analogue using themethods of the invention.

[0066] Amino acids constituting a natural amino acid residue may beselected from the 20 natural amino acids, namely glycine, alanine,valine, leucine, isoleucine, serine, threonine, aspartic acid, glutamicacid, asparagine, glutamine, lysine, arginine, cysteine, methionine,phenylalanine, tyrosine, tryptophan, histidine and proline, thatconstitute the amino acid sequence of a polypeptide of interest.

[0067] Aminoacyl-tRNA synthetases

[0068] Aminoacyl-tRNA synthetases can be from any source whethernatural, synthetic, semi-synthetic or recombinant (mutated orgenetically engineered). Accordingly, the aminoacyl-tRNA synthetases canbe from any eukaryotic or prokaryotic cell. Aminoacyl-tRNA synthetasescan have originated from the same or different cell as the host cell.Types of aminoacyl-tRNA synthetases can include but are not limited toglycine, alanine, valine, leucine, isoleucine, serine, threonine,aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine,cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine andproline t-RNA synthetases. In accordance with the invention, selectionof an appropriate aminoacyl-tRNA synthetase depends on the natural aminoacid so selected to be replaced by an amino acid analogue. For example,if an amino acid analogue will replace methionine, then a methionyl tRNAsynthetase is used.

[0069] It may be possible to use genetically engineered aminoacyl-tRNAsynthetases that recognize amino acid analogues and are able tofacilitate the incorporation of that amino acid analogue into apolypeptide. For example, hydroxy acids can be incorporated to form anester linkage in place of an amide linkage of polypeptides.

[0070] Aminoacyl-tRNA synthetases can be mutated or geneticallyengineered to enhance properties of the enzyme to facilitate theincorporation of the amino acid analogues into polypeptides of interest.For example, the editing function of the aminoacyl-tRNA synthetases canbe eliminated.

[0071] Nucleic acid sequences encoding the appropriate aminoacyl-tRNAsynthetase are used in the methods of the invention.

[0072] Amino Acid Analogues

[0073] The amino acid analogues incorporated into polypeptides using themethods of this invention are different from the twenty naturallyoccurring counterparts in their side chain functionality. The amino acidanalogue can be a close analogue of one of the twenty natural aminoacids, or it can introduce a completely new functionality and chemistry.The amino acid analogue can replace an existing amino acid in a protein(substitution).

[0074] There may be a variety of amino acid analogues that can be addedto a medium according to the present invention. Suitable amino acidanalogues include, but are not limited to, molecules having fluorinated,electroactive, conjugated, azido, carbonyl, alkyl and unsaturated sidechain functionalities. The following are representative examples ofamino acid analogues:

[0075] Amino acid analogues which are modifications of natural aminoacids in the side chain functionality, such that the imino groups ordivalent non-carbon atoms such as oxygen or sulfur of the side chain ofthe natural amino acids have been substituted by methylene groups, or,alternatively, amino groups, hydroxyl groups or thiol groups have beensubstituted by methyl groups, olefin, or azido groups, so as toeliminate their ability to form hydrogen bonds, or to enhance theirhydrophobic properties (e.g. methionine to norleucine).

[0076] Amino acid analogues which are modifications of natural aminoacids in the side chain functionality, such that the methylene groups ofthe side chain of the natural amino acids have been substituted by iminogroups or divalent non-carbon atoms or, alternatively, methyl groupshave been substituted by amino groups, hydroxyl groups or thiol groups,so as to add ability to form hydrogen bonds or to reduce theirhydrophobic properties (e.g. leucine to 2-aminoethylcysteine, orisolecine to o-methylthreonine).

[0077] Amino acid analogues which are modifications of natural aminoacids in the side chain functionality, such that a methylene group ormethyl groups have been added to the side chain of the natural aminoacids to enhance their hydrophobic properties (e.g. Leucine togamma-Methylleucine, Valine to beta-Methylvaline (t-Leucine)).

[0078] Amino acid analogues which are modifications of natural aminoacids in the side chain functionality, such that a methylene groups ormethyl groups of the side chain of the natural amino acids have beenremoved to reduce their hydrophobic properties (e.g. Isoleucine toNorvaline).

[0079] Amino acid analogues which are modifications of natural aminoacids in the side chain functionality, such that the amino groups,hydroxyl groups or thiol groups of the side chain of the natural aminoacids have been removed or methylated to eliminate their ability to formhydrogen bonds (e.g.Threonine to o-methylthreonine or Lysine toNorleucine).

[0080] Optical isomers of the side chains of natural amino acids (e.g.Isoleucine to Alloisoleucine);

[0081] Amino acid analogues which are modifications of natural aminoacids in the side chain functionality, such that the substituent groupshave been introduced as side chains to the natural amino acids (e.g.Asparagine to beta-fluoroasparagine).

[0082] Amino acid analogues which are modifications of natural aminoacids where the atoms of aromatic side chains of the natural amino acidshave been replaced to change the hydrophobic properties, electricalcharge, fluorescent spectrum or reactivity (e.g. Phenylalanine toPyridylalanine, Tyrosine to p-Aminophenylalanine).

[0083] Amino acid analogues which are modifications of natural aminoacids where the rings of aromatic side chains of the natural amino acidshave been expanded or opened so as to change hydrophobic properties,electrical charge, fluorescent spectrum or reactivity (e.g.Phenylalanine.to Naphthylalanine, Phenylalanine to Pyrenylalanine).

[0084] Amino acid analogues which are modifications of the natural aminoacids in which the side chains of the natural amino acids have beenoxidized or reduced so as to add or remove double bonds (e.g. Alanine toDehydroalanine, Isoleucine to Beta-methylenenorvaline).

[0085] Amino acid analogues which are modifications of proline in whichthe five-membered ring of proline has been opened or, additionally,substituent groups have been introduced (e.g. Proline toN-methylalanine).

[0086] Amino acid analogues which are modifications of natural aminoacids in the side chain functionality, in which the second substituentgroup has been introduced at the alpha-position (e.g. Lysine toalpha-difluoromethyllysine).

[0087] Amino acid analogues which are combinations of one or morealterations, as described supra (e.g. Tyrosine top-Methoxy-m-hydroxyphenylalanine).

[0088] Amino acid analogues which differ in chemical structures fromnatural amino acids but can serve as substrates for aminoacyl-tRNAsynthetase by assuming a conformation analogous to natural amino acidswhen bound to this enzyme. (e.g. Isoleucine to Furanomycin)

[0089] Types of amino acid analogues of methionine are6,6,6-trifluoromethionine, homoallyglycine, homoproparglycine,norvaline, norleucine, cis-crotylglycine, trans-crotylglycine,2-aminoheptanoic acid, 2-butynylglycine, allylglycine, azidoalanine andazidohomoalanine.

[0090] Vectors

[0091] In accordance with the methods of the invention, suitableexpression vectors which may be used include, but are not limited to,viral particles, baculovirus, phage, plasmids, phagemids, cosmids,phosmids, bacterial artificial chromosomes, viral DNA (e.g. vaccinia,adenovirus, foul pox virus, pseudorabies and derivatives of SV40),P1-based artificial chromosomes, yeast plasmids, yeast artificialchromosomes, and any other vectors specific for specific hosts ofinterest (such as bacillus, aspergillus, yeast, etc.) Such vectors canbe chromosomal, nonchromosomal or synthetic DNA sequences.

[0092] Large numbers of suitable vectors are known to those of skill inthe art, and are commercially available. The following vectors areprovided by way of example; Bacterial: pQE70, pQE60, pQE-9, pQE15(Qiagen, Valencia, Calif.), psiX174, pBluescript SK, pBluescript KS,(Stratagene, La Jolla, Calif.); pTRC99a, pKK223-3, pKK233-3, pDR540,pRIT2T (Pharmacia, Uppsala, Sweden); Eukaryotic: pWLNEO, pXT1, pSG(Stratagene, La Jolla, Calif.) pSVK3, pBPV, PMSG, pSVLSV40 (Pharmacia,Uppsala, Sweden).

[0093] A preferred vector for expression may be an autonomouslyreplicating vector comprising a replicon that directs the replication ofthe nucleic acids within the appropriate host cell. The preferredvectors also include an expression control element, such as a promotersequence, which enables transcription of the inserted sequences and canbe used for regulating the expression (e.g., transcription and/ortranslation) of an operably linked sequence in an appropriate host cellsuch as Escherichia coli. Methods for generating vectors are well knownin the art, for example, see Maniatis, T., et al., 1989 MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., incorporated by reference herein.

[0094] Expression control elements are known in the art and include, butare not limited to, inducible promoters, constitutive promoters,secretion signals, enhancers, transcription terminators, and othertranscriptional regulatory elements. Other expression control elementsthat are involved in translation are known in the art, and include theShine-Dalgarno sequence, and initiation and termination codons. Thepreferred vector also includes at least one selectable marker gene thatencodes a gene product that confers drug resistance, such as resistanceto ampicillin or tetracyline. The vector also comprises multipleendonuclease restriction sites that enable convenient insertion ofexogenous DNA sequences.

[0095] The preferred vectors for generating polypeptides of interest arethose compatible to prokaryotic host cells. Prokaryotic cell expressionvectors are well known in the art and are available from severalcommercial sources. For example, a pQE vector (e.g., pQE15, availablefrom Qiagen Corp., Valencia, Calif.) may be used to express polypeptidesof interest, containing natural amino acids and modified polypeptides,including those containing amino acid analogues, in bacterial hostcells.

[0096] The nucleic acids derived from a microorganism(s) may be insertedinto the vector by a variety of procedures. In general, the nucleicacids can be inserted into an appropriate restriction endonucleasesite(s) by procedures known in the art. Such procedures and others aredeemed to be within the scope of those skilled in the art.

[0097] The nucleic acid sequence encoding the aminoacyl-tRNA synthetaseor polypeptide of interest in the expression vector may be operativelylinlked to an appropriate expression control sequence(s) (promoter) todirect mRNA synthesis. Bacterial promoters include lac, lacZ, T3, T7,gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus, and mouse metallothionein-I.

[0098] Selection of the appropriate vector and its correlative promoteris well within the level of ordinary skill in the art. The expressionvector may also contain a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression. Promoter regions can beselected from any desired gene using CAT (chloramphenicol transferase)vectors or other vectors with selectable markers. Chemical ortemperature sensitive promotors can be used for inducing the expressionof either the aminoacyl-tRNA synthetase or the target protein.

[0099] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells, such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or tetracycline or ampicillinresistance in E. coli.

[0100] Inducers

[0101] In accordance with the methods of the invention when theexpression control element is an inducible promotor, the promoter may beinduced by an external stimulus, such as by adding a compound (e.g.IPTG) or by heat shocking to initiate the expression of the gene.

[0102] Level of Expression

[0103] In accordance with the methods of the invention, the productionof the aminoacyl-tRNA synthetase and/or the polypeptide of interest ispreferably in an amount in excess of the level (any increase that ismeaningful or confers a benefit) produced by a naturally occurring geneencoding the aminoacyl-tRNA synthetase and/or the polypeptide ofinterest.

[0104] The increase in the level of aminoacyl-tRNA synthetase and/or thepolypeptide of interest can be measured by monitoring an increase inprotein expression by gel electrophoresis, western blot analysis, orother relevant methods of protein detection (Maniatis, T., et al., 1989Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

[0105] The increase in the level of aminoacyl-tRNA synthetase can alsobe determined by measuring the ATP-PP₁ exchange activity (Mellot, P.;Mechulam, Y.; LeCorre, D.; Blanquet, S.; Fayat, G. J. Mol. Biol. 1989,208, 429; Blanquet, S.; Fayat, G.; Waller, J.-P. Eur. J. Biochem. 1974,44, 343; Ghosh, G.; Pelka, H.; Schulman, L. H. Biochemistry 1990, 29,2220) of cell lysates.

[0106] Fusion Genes

[0107] In accordance with the methods of the invention, a fusion geneincludes a sequence encoding a polypeptide of the invention operativelyfused (e.g., linked) to a non-related sequence such as, for example, atag sequence to facilitate isolation and/or purification of theexpressed gene product (Kroll, D. J., et al., 1993 DNA Cell Biol12:441-53). The pQE expression vectors used in this invention expressproteins fused to a poly-Histidine tag that facilitates isolation and/orpurification of the expressed gene.

[0108] Host Cells

[0109] In accordance with the methods of the invention, types of hostcells include, but are not limited to, bacterial cells, such as E. coli,Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells suchas CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. Theselection of an appropriate host is deemed to be within the scope ofthose skilled in the art from the teachings herein.

[0110] A preferred embodiment of a host cell is an auxotroph. Auxotrophsdepend upon the external environment to supply certain amino acids, forexample, a methionine auxotroph depends on methionine in the growthmedium for its survival. The choice of auxotroph is dependent on theamino acid that is selected to be replaced by an amino acid analogue inthe target protein (e.g. if methionine is selected, then a methionineauxotroph is employed, if phenylalanine is selected, then aphenylalanine auxotroph is employed).

[0111] Suitable auxotrophs include, but are not limited to CAG18491,B834(DE3), AD494, DL41, and ML304d.

[0112] Host cells may be either wild type cells or transformants. Theterm “transformants” as used herein including products oftransformation, transfection and transduction. Preferably, thepolypeptides of interest to be produced by the cells according to thepresent invention are those which wild-type cells cannot produce. Thus,it is preferable that the cells to be used in the present invention betransformants.

[0113] Host-Vector Systems

[0114] The invention further discloses a host-vector system comprising avector or vectors having nucleic acids encoding the aminoacyl-tRNAsynthetase and polypeptide of interest.

[0115] The host-vector system is used to produce the polypeptides ofinterest. The host cell can be either prokaryotic or eukaryotic.Examples of suitable prokaryotic host cells include bacterial strainsfrom genera such as Escherichia, Bacillus, Pseudomonas, Streptococcus,and Streptomyces. Examples of suitable eukaryotic host cells include ayeast cell, a plant cell, or an animal cell, such as a mammalian cell.

[0116] Introduction of the vectors of the present invention into anappropriate cell host is accomplished by well known methods thattypically depend on the type of vector used and host system employed.For transformation of prokaryotic host cells, electroporation and salttreatment methods are typically employed, see for example, Cohen et al.,1972 Proc Acad Sci USA 69:21 10; Maniatis, T., et al., 1989 MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. Transformation of vertebrate cells with vectors byelectroporation, cationic lipid or salt treatment methods, is typicallyemployed, see, for example, Graham et al., 1973 Virol 52:456; Wigler etal., 1979 Proc NatlAcadSci USA 76:1373-76.

[0117] Successfully transformed host cells, i.e., cells that contain avector of the present invention, are identified by well-knowntechniques. For example, cells resulting from the introduction of avector of the present invention are selected and cloned to producesingle colonies. Cells from those colonies are harvested, lysed andtheir nucleic acid content examined for the presence of the vector usinga method such as that described by Southern, J Mol Biol (1975) 98:503,or Berent et al., Biotech (1985) 3:208, or the proteins produced fromthe cell are assayed via a biochemical assay or immunological methodsuch as Western blotting.

[0118] The methods of the invention in which the cloned gene isexpressed in a suitable host cell, are preferred if longer polypeptides,higher yield, or a controlled degree of amino acid analogueincorporation is desired. For example, a suitable host cell isintroduced with an expression vector having the nucleotide sequenceencoding the polypeptide of interest. The host cell is then culturedunder conditions that permit in vivo production of the desiredpolypeptide, wherein one or more naturally occurring amino acids in thedesired polypeptide are replaced with the amino acid analogue analoguesand derivatives.

[0119] A preferred embodiment provides a host-vector system comprisingthe pQE15 (Qiagen, Santa Clara, Calif.) vector having a sequenceencoding the the aminoacyl-tRNA synthetase and target polypeptide ofinvention, which is introduced along with the pREP4 (Qiagen) vector intoan appropriate auxotrophic host cell such as E. coli methionineauxotroph CAG18491 strain, which is useful, for example, for producing apolypeptide where a selected natural amino acid is replaced with a aminoacid analogue.

[0120] An embodiment of the host cells of the present invention areEscherichia coli and transformants thereof, and an example of theprotein to be produced is dihydrofolate reductase.

[0121] Media

[0122] Suitable media for growing the host-vector systems of theinvention are well known in the art, for example, see Sambrook et al.,Molecular Cloning (1989), supra. In general, a suitable media containsall the essential nutrients for the growth of the host-vector system.The media can be supplemented with antibiotics that are selected forhost-vector system.

[0123] The media may contain all 20 natural amino acids or lack aselected natural amino acid. The media may also contain an amino acidanalogue in place of a selected natural amino acid.

[0124] Potential Uses of Modified Polypeptides

[0125] According to the present invention, it is now possible to createentirely new modified polypeptides, in which amino acid analogues, aswell as the 20 natural amino acids are used as constituents.

[0126] Modified polypeptides can be used to prepare functional drugs,antagonistic drugs or inhibitory agents. Also, using non-natural aminoacids in protein engineering expands the potential designs ofpolypeptides. Since such modified polypeptides are not natural, they maybe less susceptible to proteolytic enzymes generally present in cells.

[0127] Introduction of amino acid analogues in polypeptides may producemodified polypeptides having a variety of side chains having highlyactive chemical functional groups. The reactivity of the various typesof the functional groups introduced can be exploited to control proteinstructure and function. For example, polypeptides or proteins may beproduced that have undergone site-specific phosphorylation, methylationor addition of sugar chains. It may be possible to produce modifiedpolypeptides as derivatives analogous to specified proteins by theintroduction of amino acid analogues having functional groups to formcrosslinks so that cellular components which interact with the specifiedproteins in the cells can be detected. Modified polypeptides withincorporated fluorescent amino acid residues are useful to tracemetabolic pathways in organisms or to elucidate mechanisms of biologicalactions. It is possible to produce modified polypeptides having aminoacid analogues which differ in acid dissociation constant from naturalamino acids, so as to control properties of the polypeptides that dependon the acidity in aqueous solutions.

[0128] It is possible to introduce amino acid analogues intopolypeptides that will self-assemble so as to mimic viruses (e.g., coatproteins), muscle fibers (e.g., actin and myosin) or chromatin (e.g.,histones) so as to create supra-molecular structures having specifiedfunctions. Additionally, the supra-molecular structures can be furthermodified in a biological system to create other supra-molecularstructures having specified functions.

[0129] It may be possible to add amino acid analogues according to themethods of the invention to artificial feeds for silk worms that cansynthesize silk with the amino acid analogues. Further, it may bepossible to produce protein fibers with optical properties from modifiedpolypeptides into which amino acid analogues have been incorporated. Inthis regard, modified polypeptides with amino acid analogues havingfunctional groups to form crosslinkages can produce supra-molecularstructures with silk as supporting construction. Crosslinkages of themodified polypeptides can then produce new proteinaceous structures.Into the structures thus produced, non-natural fluorescent amino acidscan be introduced, e.g. to make biochips for photoenergy transduction.

ADVANTAGES OF THE INVENTION

[0130] The invention introduces a unique strategy that can be widelyapplied to incorporate amino acid analogues to substitute for any of theselected natural amino acid residues in polypeptides of interest. Agreater range of amino acid analogues can be employed for proteinsynthesis. In addition, modified polypeptides produced using the methodsof this invention can be produced in higher yields and with high levelsof replacement of natural amino acids.

[0131] The method of this invention changes the building blocks ofprotein synthesis, leaving the “blueprint” encoding the proteinsunchanged. The invention, therefore, permits a rapid and predictableapproach to protein design and produces modified polypeptides withsignificantly increased yields and expansion of amino acid analoguesthat can serve as substrates for polypeptide synthesis.

[0132] This method of this invention is generally applicable to a largerange of proteins, enzymes, and peptides, and is not limited by the sizeor structure of the proteins or polypeptides. Incorporation of aminoacid analogues with different functionalities, such as double bonds, canbe utilized for further chemical derivatization of the polypeptide ofinterest. Furthermore, the feasibility of incorporating amino acidanalogues using in vivo methods should allow the manipulation ofenzymes, signaling molecules, protein ligands, and may prove to be ofbroad utility in the engineering of more versatile biologicalassemblies.

[0133] The following examples are presented to illustrate the presentinvention and to assist one of ordinary skill in making and using thesame. The examples are not intended in any way to otherwise limit thescope of the invention.

Example I

[0134] This example demonstrates the selectivity of methionyl t-RNAsynthetase for methionine analogues and the efficient incorporation ofunsaturated methionine analogues into proteins in vivo.

[0135] Synthesis of Amino Acid Analogues

[0136] Each of the analogues 2-7 and 11 (FIG. 2) was prepared byalkylation of diethyl acetamidomalonate with the appropriate alkyltosylate followed by decarboxylation and deprotection of the aminefunction. This section provides information on general syntheticprocedures and a detailed protocol for preparation of 2. Similar methodswere used to prepare 3-7 and 11. Analogues 8, 9, 12, and 13 areavailable commercially (Sigma-Aldrich, St. Louis, Mo). Analogue 10 wasprepared as described by Blackwell et al (H. E. Blackwell, R. H. Grubbs,Angew. Chem. 1998, 110, 3469-3472; Angew. Chem. Int. Ed. 1998, 37,3281-3284.).

[0137] General Procedures.

[0138] Glassware was dried at 150° C. and cooled under nitrogen prior touse. Tetrahydrofuran (THF) was freshly distilled from Na/benzophenone.N,N-Dimethylformamide (DMF) was distilled and stored over BaO. Pyridine(99.8%, anhydrous, Aldrich) and other reagents and solvents were used asreceived. ¹H NMR spectra were recorded on Bruker AC 200 and AMX 500spectrometers and ¹³C NMR spectra were recorded on a Bruker DPX 300spectrometer. Column chromatography was performed with silica gel 60,230-400 mesh (EM Science); silica 60-F254 (Riedel-de Haën) was used forthin layer chromatography.

[0139] DL-2-amino-5-hexenoic Acid (2)

[0140] (Drinkwater, D. J.; Smith, P. W. G. J. Chem. Soc. C 1971, 1305;Baldwin, J. E.; Hulme, C.; Schofield, C. J. J. Chem. Res. (S) 1992,173).

[0141] 3-Buten-1-ol 4-methylbenzene Sulfonate.

[0142] A solution of 3 g (42 mmol) 3-buten-1-ol in 10 mL dry pyridinewas cooled in an ice bath. Tosyl chloride (7.9 g, 42 mmol), was added.After stirring for 3 h the mixture was poured into 30 mL of anice/concentrated HCl 4/1 v/v solution, extracted with 60 mL diethylether and dried overnight in the freezer over MgSO₄. The mixture wasfiltered and the ether evaporated to yield 7.22 g (76%) of 3-buten-1-ol4-methylbenzene sulfonate as a yellow oil. ¹H NMR (CDCl₃):δ 2.39-2.53(m, 2H, J=6.5 and 6.9 Hz, CH ₂—CH═CH₂; and s, 3H,CH ₃—Ar),4.08(t,J=6.5Hz,2H, CH ₂OSO₂),5.09-5.15(m,2H,J_(Z)═10.4,J_(E)=16.6,J_(gem)=3.1Hz, CH₂—CH═CH ₂), 5.57-5.82(m,1H,J_(Z)=10.4,J_(E)=16.6,J =6.9Hz, CH₂—CH═CH₂),7.38 and 7.72 (d,4H,J=8.6Hz, Ar).

[0143] Acetylamino-3-butenyl-propanedioic Acid Diethyl Ester.

[0144] Diethyl acetamidomalonate, 1.56 g (6.9 mmol), was dissolved atroom temperature under N₂ in 10 mL dry THF. Potassium tert-butoxide(0.80 g, 7 mmol), was added under vigorous stirring. The mixture washeated for 2 h at 60° C. 3-Buten-1-ol 4-methylbenzenesulfonate (1.5 g,6.9 mmol) was added, and the mixture was heated under reflux for 2 days.The THF was removed, the residue was quenched with 10 mL 1 M HCl, andthe crude product was extracted with ethyl acetate (25 mL). The ethylacetate solution was washed twice with 25 mL water, dried over MgSO₄,filtered and concentrated. The crude product was purified by columnchromatography (eluent cyclohexane/ethyl acetate 2/1 v/v) to yield 0.82g (44%) of acetylamino-3-butenyl-propanedioic acid diethyl ester. ¹H NMR(CDCl₃): δ 1.28(t,6H, J=7.2Hz, CH₃ —CH₂), 1.78-2.0 (m, 2H,J=8.3, 6.5Hz,CH₂═CH—CH ₂—CH₂),2.08(s,3H,CONH—CH ₃),2,45(m, 2H,J=8.3Hz,CH₂═CH—CH₂—CH₂),4.25(q,4H, J=7.2Hz, CH₃—CH ₂),4,90-5.09(m,2H,J_(z)=10.4,J_(E)=16.6,J_(gem)=3.2Hz, CH₂—CH═CH ₂), 5.61-5.90 (m,1H,J_(z)=10.4,J_(E)=16.6,J=6.5Hz,CH₂—CH═CH₂),6.78(s,1H, CONH—CH₃).

[0145] DL-2-amino-5-hexenoic Acid.

[0146] The diethyl ester obtained as described above was hydrolyzed tothe dicarboxylate by heating under reflux for 4 h in 25 mL 10 w % NaOH.The solution was neutralized with 6 M HCl and the solvent wasevaporated. The diacid was extracted with 25 mL of methanol. Followingsolvent evaporation, 20 mL 1M HCl was added and the solution wasrefluxed for 3 h. The solvent was evaporated and the product was takenup in 10 mL methanol. Propylene oxide (5 mL) was added and the mixturewas stirred overnight at room temperature. The precipitate was filteredand dried, yielding DL-2-amino-5-hexenoic acid (0.47 g, 63%). Theproduct was recrystallized from EtOH/H₂O 2/1 v/v (0.28 g, 60%). The ¹HNMR data were in agreement with those of reference 16 (Hatanaka, S.-I.;Furukawa, J.; Aoki, T.; Akatsuka, H.; Nagasawa, E. Mycoscience, 1994,35,391). ¹H NMR (D₂O):δ 1.78-2.0(m,2H, J=6.4, 6.6Hz,CH₂═CH—CH ₂—CH₂),2.08-2.20(m,2H,J=6.1, 6.4Hz, CH₂═CH—CH₂—CH ₂),3.75(t,1H,J=6.1Hz,H₂N—CH—COOH), 4.90-5.12(m,2H,J_(z)=10.5,J_(E)=16.7,J_(gem)=3.3Hz, CH₂—CH═CH ₂),5.61-5.90(m,1H,J_(z)=10.5,J_(E)=16.7,J=6.6Hz, CH₂—CH═CH₂). ¹³C NMR(D₂O):δ 28.9 (CH₂═CH—CH₂—CH₂),29.9(CH₂═CH—CH₂—CH₂),54.4(H₂N—CH—COOH),116.3 (CH₂—CH═CH₂),137.3(CH₂—CH═CH₂),174.8(COOH).

[0147] Determination of Translational Activity

[0148] Buffers and media were prepared according to standard protocols(Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning. ALaboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. 1989; Ausubel, F. M.; Brent, K.; Kingston, K. E.;Moore, D. D.; Scidman, J. G.; Smith, J. A.; Struhl, K. Current Protocolsin Molecular Biology, John Wiley & Sons, NY 1995). The E. colimethionine auxotroph CAG18491 (λ, rph-1, metE3079.:Tn10) (obtained fromthe Yale E. coli Genetic Stock Center), was transformed with plasmidspREP4 and pQE15 (Qiagen, Valencia, Calif.), to obtain the expressionhost CAG18491/pQE15/pREP4.

[0149] Protein Expression (5 mL Scale).

[0150] M9AA medium (50 mL) supplemented with 1 mM MgSO₄, 0.2 wt %glucose, 1 mg/L thiamine chloride and the antibiotics ampicillin (200mg/L) and kanamycin (25 mg/L) was inoculated with 2 mL of an overnightculture of CAG18491/pQE15/pREP4. When the turbidity of the culturereached an optical density at 600 nm (OD₆₀₀) of 0.8, a medium shift wasperformed. The cells were sedimented for 10 min at 3030 g at 4° C., thesupernatant was removed, and the cell pellet was washed twice with 20 mLof M9 medium. Cells were resuspended in 50 mL of the M9AA mediumdescribed above, without methionine. Test tubes containing 5 mL aliquotsof the resulting culture were prepared, and were supplemented with 200tμL 1 mg/mL (0.27 mM) L-methionine (1) (positive control),DL-2-amino-5-hexenoic acid (2) (0.31 mM), DL-homopropargylglycine(3)(0.31 mM), cis-or trans-DL-2-amino-4-hexenoic acid (4 or 5) (0.31 mM),DL-6,6,6-trifluoro-2-amino hexanoic acid (6) (0.22 mM),DL-2-aminoheptanoic acid (7) (0.28 mM), L-norvaline (8) (0.34 mM) orL-norleucine (9) (0.31 mM), respectively. A culture lacking methionine(or any analogue) served as the negative control. Protein expression wasinduced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to afinal concentration of 0.4 mM. Samples were taken every hour for 4 h,the OD₆₀₀ was measured, and the samples were sedimented. After thesupernatant was decanted, the cell pellets were resuspended in 20 μLdistilled H₂O. Protein expression was monitored by SDS polyacrylamidegel electrophoresis (12% acrylamide running gel, 12 mA, 14 h), using anormalized OD₆₀₀ of 0.2 per sample.

[0151] Protein Expression (1 L Scale)

[0152] Similar procedures were used for preparation and isolation ofmDHFR from media supplemented with 1, 2 or 3. The example presented isfor medium supplemented with 3. M9AA medium (100 mL) supplemented with 1mM MgSO₄ 0.2 w % glucose, 1 mg/L thiamine chloride and the antibioticsampicillin (200 mg/L) and kanamycin (25 mg/L) was inoculated with E.coli strain CAG18491/pQE15/pREP4 and grown overnight at 37° C. Thisculture was used to inoculate 900 mL M9AA medium supplemented asdescribed. The cells were grown to an OD₆₀₀ of 0.94 and the medium shiftwas performed as described for the small scale experiments, followed byaddition of 40 mL of 1 mg/mL DL-homopropargylglycine (3). IPTG (0.4 mM)was added, and samples were taken at 1 hour intervals. OD₆₀₀ wasmeasured, the samples were sedimented and decanted, and the cell pelletswere resuspended in 20 μL distilled H₂O. Protein expression wasmonitored by SDS polyacrylamide gel electrophoresis (12% acrylamiderunning gel, 12 mA, 15 h).

[0153] Protein Purification

[0154] Approximately 4.5 h after induction, cells were sedimented (9,800g, 10 min, 4° C.) and the supernatant was removed. The pellet was placedin the freezer overnight. The cells were thawed for 30 min at 37° C., 30mL of buffer (6 M guanidine-HCl, 0.1 M NaH₂PO₄, 0.01 M Tris, pH 8) wasadded and the mixture was shaken at room temperature for 1 h. The celldebris was sedimented (15,300 g, 20 min, 4° C.) and the supernatant wassubjected to immobilized metal affinity chromatography (Ni-NTA resin)according to the procedure described by Qiagen (The QiagenExpressionist, Purification Procedure 7, 1992, 45). The supernatant wasloaded on 10 mL of resin which was then washed with 50 mL of guanidinebuffer followed by 25 mL of urea buffer (8 M urea, 0.1 M NaH₂PO₄ and0.01 M Tris, pH 8). Similar urea buffers were used for three successive25 mL washes at pH values of 6.3, 5.9 and 4.5, respectively. Targetprotein was obtained in washes at pH 5.9 and 4.5. These washes werecombined and dialyzed (Spectra/Por membrane 1, MWCO=6-8 kDa) againstrunning distilled water for 4 days, followed by batchwise dialysisagainst doubly distilled water for one day. The dialysate waslyophilized to yield 70 mg of modified mDHFR (mDHFR-Y). A similarprocedure using medium supplemented with 2 yielded 8 mg of mDHFR-E. Acontrol experiment in 2xYT medium afforded 60 mg of mDHFR. Amino acidanalyses, electrospray mass spectrometry and N-terminal proteinsequencing was performed on the mDHFR isolated.

[0155] Enzyme Purification and Activation Assays

[0156] The fully active, truncated form of methionyl tRNA synthetase(MetRS) was purified from overnight cultures of JM 101 cells carryingthe plasmid pGG3. (The plasmid, which encodes the tryptic fragment ofMetRS, Ghosh, G.; Brunie, S.; Schulman, L. H. J. Biol. Chem. 1991, 266,17136-17141). The enzyme was purified by size exclusion chromotographyas previously described (Mellot, P.; Mechulam, Y.; LeCorre, D.;Blanquet, S.; Fayat, G. J. Mol. Biol. 1989, 208, 429). Activation ofmethionine analogues by MetRS was assayed via the amino-acid-dependentATP-PP₁ exchange reaction, also as previously described (Mellot, P.;Mechulam, Y.; LeCorre, D.; Blanquet, S.; Fayat, G. J. Mol. Biol. 1989,208, 429; Blanquet, S.; Fayat, G.; Waller, J.-P. Eur. J. Biochem. 1974,44, 343; Ghosh, G.; Pelka, H.; Schulman, L. H. Biochemistry 1990, 29,2220). The assay, which measures the ³²P-radiolabeled ATP formed by theenzyme-catalyzed exchange of ³²P-pyrophosphate (PP₁) into ATP, wasconducted in 150 μl of reaction buffer (pH 7.6, 20 mM imidazole, 0.1 mMEDTA, 10 mM β-mercaptoethanol, 7 mM MgCl₂, 2 mM ATP, 0.1 mg/ml BSA, and2 mM PP₁ (in the form of sodium pyrophosphate with a specific activityof approximately 0.18 TBq/mole)). Assays to determine if the methionineanalogues 2-13 are recognized by MetRS were conducted in solutions 75 nMin enzyme and 5 mM in the L-isomer of the analogue with a reaction timeof 20 minutes. Kinetic parameters for analogue 5 were obtained with anenzyme concentration of 75 nM and analogue concentrations of 100 μM to10 mM. Parameters for methionine were obtained by using concentrationsranging from 10 μM to 1 mM. K_(m) values for methionine matched thosepreviously reported (Ghosh, G.; Pelka, H.; Schulman, L. H.; Brunie, S.Biochemistry 1991, 30, 9569), though the measured kcat was somewhatlower than the literature value. Aliquots of 20 μl were removed from thereaction mixture at various time points and were quenched in 0.5 ml of asolution comprising 200 mM PP₁, 7% w/v HClo₄, and 3% w/v activatedcharcoal. The charcoal was rinsed twice with 0.5 mL of a 10 mM PP₁, 0.5%HClO₄ solution and was then resuspended in 0.5 mL of this solution andcounted via liquid scintillation methods. Kinetic constants werecalculated by nonlinear regression analysis.

[0157] Computation

[0158] Single-point energy ab initio calculations (Hartree-Fock model,6-31 G* basis set) (Hehre, W. J.; Ditchfield, R.; Pople, J. A. J. Chem.Phys. 1972, 56, 2257; Hariharan, P. C.; Pople, J. A. Chem. Phys. Lett.1972, 66, 217; Francl, M. M. et al. J. Chem. Phys. 1982, 77, 3654) wereperformed for methionine and for analogues 2, 3 and 5 with fullyextended side chains. Electron density maps are shown as surfaces ofelectron density 0.08 electrons/au³. Isopotential plots are representedas surfaces where the energy of interaction between the amino acid and apoint positive charge is equal to −10 kcal/mole. Calculations wereperformed by using the program MacSpartan (Wavefunction, Inc., Irvine,Calif., USA).

[0159] Results and Discussion

[0160] Methionine Analogues

[0161] Methionine analogues 2-13 were investigated with respect to theircapacity to support protein synthesis in E. coli cells depleted ofmethionine. Norvaline (8) and norleucine (9), allylgycine (12), andpropargylglycine (13) are commercially available. Analogues 2-7 and 11were prepared by alkylation of diethyl acetamidomalonate with thecorresponding tosylates via standard procedures, and the remaininganalogues were prepared as described supra. In the cases of the cis- andtrans-crotylglycines (4 and 5) the tosylates were prepared in situ, andbecause of fast exchange of the tosyl group with chloride ion, mixturesof the chloride and the tosylate were obtained. Hydrolysis of themalonate and conversion to the amino acid had to be performed under mildacidic conditions for analogues 2,4 and 5; treatment with 6 N HCl, orreflux in 1 N HCl for more than 5 h led to HCl addition to the doublebond. In all cases the analogues were obtained as racemates and wereused as such.

[0162] Protein Expression

[0163]E. coli strain CAG18491/pQE15/pREP4, which produces the testprotein mDHFR upon induction with IPTG, was used as the expression host.The parent strain CAG18491 is dependent on methionine for growth, owingto insertion of transposon Tn10 into the metE gene, which is essentialfor the final step in the endogenous synthesis of methionine. Cultureswere grown in minimal medium supplemented with methionine until a celldensity corresponding to OD₆₀₀ 0.8-1.0 was reached. Cells weresedimented, washed and resuspended in minimal medium without methionine.Aliquots of the culture were then supplemented with one of the analogues2-13. Protein synthesis was induced with IPTG and cell growth andprotein expression were followed over a 4 h period. Expression resultsare presented in FIG. 3, and show clearly that analogues 2 and 3 exhibittranslational activity sufficient to allow protein synthesis in theabsence of methionine. Analogues 4-8 and 12-13 are not active in theassay reported here, while the known translational activity ofnorleucine (9) was confirmed. CAG 18491/pQE15/pREP4cultures did not growin minimal media in which methionine was replaced by 2 and 3, at thetime of inoculation.

[0164] Analysis ofProtein Structure

[0165] The extent of replacement of methionine by analogues 2 and 3 wasdetermined by an amino acid analysis, N-terminal sequencing, and (for 2)¹H nuclear magnetic resonance spectroscopy (Table 1). Proteinscontaining 2 and 3 were designated mDHFR-E (alkene) and mDHFR-Y(alkyne), respectively. TABLE 1 Protein Yield and Extent of MethionineReplacement Yield Replacement (%) Protein (mg)^(a) Amino Acid AnalysisSequencing ¹H NMR mDHFR-E  8  86 92 77 mDHFR-Y 70 100 88 Not determined

[0166] mDHFR-E

[0167] Amino acid analysis of mDHFR-E showed a methionine content of 0.5mol % vs. the value of 3.8 mol % expected for mDHFR. Although 2 appearsto be unstable under the conditions used to hydrolyze the protein foramino acid analysis, assumption that the decrement in methionine contentis due to replacement by 2 affords an estimate of 86% substitution bythe analogue. This estimate is consistent with the results of N-terminalsequencing of mDHFR-E (FIG. 4), which indicates 92% occupancy of theinitiator site by 2. In the chromatograms shown in FIG. 4, the signaldue to methionine appears at a retention time of 12.3 min, while thatfrom 2 elutes at 14.3 min. The retention time of the signal arising from2 was verified by analysis of an authentic sample of the analogue.Retention of the N-terminal residue in mDHFR was expected on the basisof the known correlation between the extent of methionine excision fromE. coli proteins and the identity of the penultimate amino acid residue(Hirel, P. H.; Schmitter, J. M.; Dessen, P.; Fayat, G.; Blanquet, S.Proc. Natl Acad. Sci. USA 1989, 86, 8247). Finally, direct evidence forincorporation of the alkene function of 2 was obtained from ¹H NMRspectroscopy. The vinyl CH resonance of 2 appears at a chemical shift of5.7 ppm in the spectrum of mDHFR-E, and can be integrated to yield anestimate of 77% replacement of methionine by the unsaturated analogue. Ayield of 8 mg of mDHFR-E was obtained from a 1 L culture of CAG18491/pQE15/pREP4grown in M9AA medium supplemented with 2, compared with70 mg obtained from a similar experiment in medium supplemented withmethionine.

[0168] mDHFR-Y.

[0169] Methionine could not be detected via amino acid analysis ofmDHFR-Y, suggesting quantitative replacement of methionine by the alkyneanalogue 3. N-terminal sequencing (FIG. 4) indicated 88% occupancy ofthe initiator site by 3. ¹H NMR analysis of mDHFR-Y was consistent withnear-quantitative replacement of methionine, as the thiomethyl resonanceat 2.05 ppm—which is prominent in the spectrum of mDHFR—could not bedetected. New signals at 2.2-2.3 ppm—which are not observed in thespectrum of mDHFR and which correspond to signals due to the β-andε-protons of 3-appeared in the spectrum of mDHFR-Y, but were notintegrated carefully owing to overlap with neighboring resonances. Theyield of mDHFR-Y obtained from M9AA medium supplemented with 3 wasessentially identical to that of mDHFR isolated from media supplementedwith methionine.

[0170] Enzyme Assays

[0171] The relative rates of activation of methionine and methionineanalogues 2-13 by MetRS were estimated by the ATP-PP_(i) exchange assay.The results shown in FIG. 5 illustrate the amount of PP₁ exchanged at areaction time of 20 minutes under standard assay conditions (seeExperimental Section). Methionine (1) is activated most efficiently bythe enzyme, causing exchange of 9 nmoles of PP_(i) over the time courseof the reaction. Analogues 2 and 3 cause exchange of PP₁ at ratessimilar to that of norleucine (9), while the remaining analogues 4, 6-8,and 12-13 cause exchange of PP_(i) at levels no higher than background(FIG. 5, lane14). The background (lane 14) is given for a reactionmixture lacking both the enzyme and the amino acid. Although analogues 5and 11 effect very slow exchange of PP_(i), the activation rate isapparently too low to support protein synthesis at a level that isdetectable in the in vivo assays. Kinetic parameters were determined formethionine and 5 as outlined in the Experimental Section. Comparison ofthe k_(cat)/K_(m) values obtained for methionine (0.54ε⁻¹ μM⁻¹) and 5(1.1×10³¹ ⁴s³¹ ¹ μM⁻¹) show that 5 is activated 4700-fold lessefficiently than methionine by MetRS. Comparison of the k_(cat)/K_(m)values obtained for methionine (0.54s⁻¹ μM⁻¹) and 11 (3.9×10⁻⁵s⁻¹ μM⁻¹)show that 11 is activated 13825-fold less efficiently than methionine byMetRS.

[0172] Discussion

[0173] A bacterial host strain (designated CAG18491/pQE15/pREP4)suitable for testing the translational activity of methionine analogues2-8 and 10-13 was prepared by transformation of E. coli strain CAG18491,a methionine auxotroph, with the repressor plasmid pREP4 and theexpression plasmid pQE15. pQE15 encodes mouse dihydrofolate reductase(mDHFR) under control of a bacteriophage T5 promoter, and appends tomDHFR an N-terminal hexahistidine sequence that facilitates purificationof the protein by immobilized metal affinity chromatography. mDHFRcontains eight methionine residues, each a potential site forsubstitution by analogues 2-8 and 10-13. The translational activity ofeach analogue was assayed on the basis of its capacity to supportsynthesis of mDHFR in cultures of CAG18491/pQE15/pREP4 that had beendepleted of methionine. In those instances in which the test protein wasdetected by gel electrophoresis (i.e., for 2 and 3), the modified mDHFRwas purified and analyzed to determine the extent of methioninereplacement by the analogue.

[0174] The results of the in vivo assays illustrated in FIG. 3 showclearly that homoallylglycine (2) and homopropargylglycine (3) serveeffectively as methionine surrogates in bacterial protein synthesis. Incontrast, analogues 4-8 and 10-13 do not support measurable levels ofprotein synthesis in bacterial cultures depleted of methionine. It ishighly unlikely that recognition by the elongation factors of theribosome or transport into the cell are the limiting factors forincorporation of these analogues. The ribosome is remarkably permissivetoward amino acid analogues with widely varying chemical functionality,as has been demonstrated by the numerous analogues incorporated intoproteins in in vitro translation experiments (Cornish, V. W.; Mendel,D.; Schultz, P. G. Angew. Chem. Int. Ed. Engl. 1995, 34, 621; Robertson,S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722;Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G.Science 1989, 244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.;Chamberlin, A. R. J. Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al.Nature 1992, 356, 537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A.Chem. Biol. 1997, 4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271,19991; Nowak, M. W. et al. Science, 1995, 268, 439; Saks, M. E. et al.J. Biol. Chem. 1996, 271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc.1999, 121, 34).

[0175] Transport of several analogues into the cell is indicated by anumber of literature reports. Analogue 4 is an antagonist formethionine, inhibiting the growth of E. coli cells (Skinner, C. G.;Edelson, J.; Shive, W. J. Am. Chem. Soc. 1961, 83, 2281); 5 has beenincorporated into proteins in E. coli cells with appropriatelyengineered MetRS activity; and 8 replaces leucine in human hemoglobinexpressed in E. coli (Apostol, I.; Levine, J.; Lippincott, J.; Leach,J.; Hess, E.; Glascock, C. B.; Weickert, M. J.; Blackmore, R. J. Biol.Chem. 1997, 272, 28980). Although there is no similar evidence reportedfor analogues 6 and 7, the fact that trifluoromethionine and ethionineare incorporated into proteins expressed in E. coli (Hendrickson, W. A.;Horton, J. R.; Lemaster, D. M. EMBO J. 1990, 9, 1665; Boles, J. O. et alNature Struct. Biol. 1994, 1, 283; Cowie, D. B.; Cohen, G. N.; Bolton,E. T.; de Robichon-Szulmajster, H. Biochim. Biophys. Acta 1959, 34, 39;Duewel, H.; Daub, E.; Robinson, R.; Honek, J. F. Biochemistry 1997, 36,3404; Budisa, N.; Steipe, B.; Demange, P.; Eckerskorn, C.; Kellerman,J.; Huber, R. Eur. J. Biochem. 1995, 230, 788) suggests that neither thetrifluoromethyl group nor the longer side chain will inhibit transportof analogues 6 and 7 into E. coli cells.

[0176] The results of the in vitro enzyme assays shown in FIG. 5 areconsistent with the in vivo results, as the analogues that support thehighest rates of PP₁ exchange also support protein synthesis in theabsence of methionine. Although the in vitro results indicate that 5 and11 are recognized by MetRS, comparison of the kcat/Km values ofmethionine and 5 and 11, demonstrate that 5 is activated 4700-fold and11 13825-fold less efficiently than methionine; thus it is notsurprising that neither 5 or 11 support measurable protein synthesis inthe in vivo experiments. Consideration of the in vivo and in vitroresults, along with the reports cited earlier, suggests that transportis not limiting and that analogue incorporation is controlled by theMetRS.

[0177] Although the crystal structure of an active tryptic fragment ofthe E. coli MetRS (complexed with ATP) has been reported (Brunie, S.;Zelwer, C.; Risler, J. L. J. Mol. Biol. 1990, 216, 411; Mechulam, Y.;Schmitt, E.; Maveyraud, L.; Zelwer, C.; Nureki, O.; Yokoyama, S.; Konno,M.; Blanquet, S. J. Mol. Biol. 1999, 294, 1287-1297), the correspondingstructure with bound methionine is not yet available. Inferencesconcerning the mechanism of methionine (or analogue) recognition byMetRS have heretofore been made indirectly, on the basis of sequencecomparison and site-directed mutagenesis (Ghosh, G.; Pelka, H.;Schulman, L. H.; Brunie, S. Biochemistry 1991, 30, 9569; Fourmy, D.;Mechulam, Y.; Brunie, S.; Blanquet, S.; Fayat, G. FEBS Lett. 1991, 292,259; Kim, H. Y.; Ghosh, G.; Schulman, L. H.; Brunie, S.; Jakubowski, H.Proc. Natl. Acad. Sci. USA 1993,90, 11553).

[0178]FIG. 6 compares the equipotential surfaces calculated formethionine and for analogues 2, 3 and 5. That 2 might serve as asubstrate for the methionyl-tRNA synthetase is not surprising, given thesimilar geometries accessible to 1 and 2, the availability ofπ-electrons near the side-chain terminus of 2, and the knowntranslational activity of norcleucine (9), the saturated analogue of 2.The high translational activity observed for 3, (i.e. near-quantitativereplacement of methionine without loss of protein yield), was notanticipated, since the colinearity of side-chain carbons 4-6 imposes on3 a geometry substantially different from that of methionine. However,the electron density associated with the triple bond of 3 is positionedsimilarly to that of the thioether of the natural substrate, despite thedifferences in side-chain geometry. Furthermore, given the importantroles assigned to residues Phe197 and Trp305 in the E. colimethionyl-tRNA synthetase (Ghosh, G.; Pelka, H.; Schulman, L. H.;Brunie, S. Biochemistry 1991, 30, 9569; Fourmy, D.; Mechulam, Y.;Brunie, S.; Blanquet, S.; Fayat, G. FEBS Lett. 1991, 292, 259; Kim, H.Y.; Ghosh, G.; Schulman, L. H.; Brunie, S.; Jakubowski, H. Proc. Natl.Acad. Sci. USA 1993, 90, 11553), alkynyl C-Hπ contacts (Steiner, T.;Starikov, E. B.; Amado, A. M.; Teixeira-Dias, J. J. C. J. Chem. Soc.Perk Trans. 2, 1995, 7, 1321) and the polarizability of the unsaturatedside chain may also play significant roles in recognition of 3 by theenzyme. FIG. 6 also compares the geometries of 1 and 5, the latter ananalogue neither recognized efficiently by the MetRS in vitro nortranslationally active in vivo. Although the geometries of 1 and 5appear similar in the representation shown, the fixed planarity of theC₄-C₅ bond may preclude the side-chain conformation required forefficient recognition of 5 by MetRS. Appropriate engineering of theMetRS activities of E. coli imparts translational activity to 5.

[0179] In conclusion, a set of twelve methionine analogues was assayedfor translational activity in Escherichia coli. Norvaline andnorleucine, which are commercially available, were assayed along withhomoallylglycine (2), homopropargylglycine (3), cis-crotylglycine (4),trans-crotylglycine (5), 6,6,6-trifluoro-2-aminohexanoic acid (6) and2-aminoheptanoic acid (7) and 2-butynylglycine (11), each of which wasprepared by alkylation of diethyl acetamidomalonate with the appropriatetosylate, followed by hydrolysis. The other analogues were commerciallyavailable or prepared as described supra. The E. coli methionineauxotroph CAG 18491, transformed with plasmids pREP4 and pQE15, was usedas the expression host, and translational activity was assayed bydetermination of the capacity of the analogue to support synthesis ofthe test protein dihydrofolate reductase (mDHFR) in the absence of addedmethionine.

[0180] The importance of amino acid side chain length was illustrated bythe fact that neither norvaline (8) nor 7 showed translational activity,in contrast to norleucine (9), which does support protein synthesisunder the assay conditions. The internal alkene functions of 4 and 5prevented incorporation of these analogues into test protein, and thefluorinated analogue 6 and 10-13 yielded no evidence of translationalactivity. The terminally-unsaturated compounds 2 and 3, however, provedto be excellent methionine surrogates: ¹H NMR spectroscopy, amino acidanalysis and N-terminal sequencing indicated ca 85% substitution ofmethionine by 2, while 3 showed 90-100% replacement. Both analogues alsofunction efficiently in the initiation step of protein synthesis, asshown by their near-quantitative occupancy of the N-terminal amino acidsite in mDHFR. Enzyme kinetics assays were conducted to determine therate of activation of each of the methionine analogues by methionyl tRNAsynthetase (MetRS); results of the in vitro assays corroborate the invivo incorporation results, suggesting that success or failure ofanalogue incorporation in vivo is controlled by MetRS.

Example II

[0181] This example demonstrates the expansion of the scope ofmethionine analogues for incorporation into proteins in vivo by alteringthe methionyl-tRNA synthetase activity of a bacterial expression host.

[0182] The relative rates of activation of methionine and methionineanalogues 2 -13 (FIG. 2) by MetRS were characterized in vitro by theATP-PPI exchange assay. The fully active, truncated form of MetRS waspurified from overnight cultures of JM101 cells carrying the plasmidpGG3. The enzyme was purified by size exclusion chromatography aspreviously described (P. Mellot, Y. Mechulam, D. LeCorre, S. Blanquet,G. Fayat, J. Mol. Biol. 1989, 208, 429-443). Activation of methionineanalogues by MetRS was assayed at 25° C. via the amino acid-dependentATP-PPI exchange reaction, also as described in Example I (G. Ghosh, H.Pelka, L. H. Schulman, Biochemistry 1990, 29, 2220-2225). Assays todetermine if the methionine analogues 2 -13 were recognized by MetRSwere conducted in solutions 75 nM in enzyme and 5 mM in the L-isomer ofthe analogue with a reaction time of 20 minutes. Kinetic parameters foranalogue 5 were obtained with an enzyme concentration of 50 nM andanalogue concentrations of 100 μM to 10 mM. Kinetic parameters foranalogue 11 were determined using an enzyme concentration of 50 nM andanalogue concentrations ranging from 750 μM to 20 mM. Kinetic parametersfor analogues 4, 7, 8, and 12 were obtained with an enzyme concentrationof 50 or 75 nM and analogue concentrations ranging from 5 to 70 mM.Parameters for methionine were obtained by using concentrations rangingfrom 10 μM to 1 mM. Km values for methionine were similar to thosepreviously reported (24±2 μM), though the measured k_(cat) was somewhatlower than the literature value (13.5s⁻¹) (H. Y. Kim, G. Ghosh, L. H.Schulman, S. Brunie, H. Jakubowski, Proc. Natl. Acad. Sci. USA 1993, 90,11553-11557). Kinetic constants were calculated by nonlinear regressionanalysis.

[0183]FIG. 5 demonstrates that analogues 2 and 3 are activated by MetRS,as anticipated on the basis of the in vivo experiments (as described inExample I, infra; J. C. M. van Hest, D. A. Tirrell, FEBS Lett. 1998,428, 68-70), although they cause exchange of PP_(i) at ratesseveral-fold lower than methionine. Analogue 4 does not cause measurableexchange of PP_(i) by MetRS in vitro, which was expected since neither 4nor 5 were indicated to be translationally active in vivo.

[0184] Analogues 5 and 11, however, were activated by MetRS, causingslow exchange of PP_(i)under the assay conditions used herein. Table 2shows the k_(cat)/K_(m) values obtained for methionine, 2, 3, 5, 9, and11. Given that k_(cat)/K_(m) for 5 is 4700-fold and that for 11 is13825-fold lower than that for methionine (as described in Example I,infra), it is not surprising that neither 5 or 11 support measurableprotein synthesis within the time frame of the in vivo experiments.

[0185] These results suggest that increasing the MetRS activity of theexpression host might allow efficient protein synthesis in culturessupplemented with 5 or 11. This strategy was not employed previously forincorporating amino acid analogues into proteins in vivo, but reports ofin vivo misacylation of tRNA substrates by overexpressed aminoacyl-tRNAsynthetase supported the viability of the approach (S. Li, N. V. Kumar,U. Varshney, U. L. RajBhandary, J. Biol. Chem. 1996, 271, 1022-1028; J.M. Sherman, M. J. Rogers, D. Soll, Nuc. Acids. Res. 1992, 20, 2847-2852;U. Varshney, U. L. RajBhandary, J. Bacteriol. 1992, 1 74, 7819-7826; R.Swanson, P. Hoben, M. Sumner-Smith, H. Uemura, L. Watson, D. Soll,Science 1988, 242, 1548-1551). TABLE 2 K_(cat)/K_(m) values obtained formethionine, 2, 3, 5, 9, and 11 Protein Yield, Analogue K_(m) (μM)k_(cat) (s⁻¹) k_(cat)/K_(m) (s⁻¹μM⁻¹) mg/L 1  24.3 ± 2   13.3 ± 0.2 5.47× 10⁻¹ 35 3 2415 ± 170 2.60 ± 0.3 1.08 × 10⁻³ 35 9 4120 ± 900 2.15 ± 0.65.22 × 10⁻⁴ 20 2 4555 ± 200 1.35 ± 0.1 2.96 × 10⁻⁴ 10 5 15,675 ± 250  1.82 ± 0.6 1.16 × 10⁻⁴  0  11 38,650 ± 2000  1.51 ± 0.5 3.91 × 10⁻⁵  0

[0186] Generation of Host-vector System

[0187] A bacterial host capable of overexpressing MetRS was produced bytransforming E. coli strains B834(DE3) (Novagen, Inc., Madison, Wis.,USA), a methionine auxotroph, with repressor plasmid pREP4 andexpression plasmid pQE15-MRS (FIG. 19) (SEQ ID NO.: 1). A gene encodinga mutant MetRS was removed from plasmid pBSM547W305F (D. Fourmy, Y.Mechulam, S. Brunie, S. Blanquet, G. Fayat, FEBS Lett. 1991, 292,259-263) by treatment with restriction enzymes Sac I and Kpn I. The SacI/Kpn I fragment (2450 bp) was ligated into the cloning vectorpUC19-Nhelink, which was constructed to permit the cohesive ends of themutant MetRS gene to be changed to Nhe I. The MetRS gene with Nhe Icohesive ends was then ligated into the unique Nhe I site of the plasmidpQE15 (Qiagen, Inc., Santa Clarita, Calif., USA) to yield plasmidpQEI5-W305F (FIG. 20) (SEQ ID NO.: 2). Transformation of pQE15-W305F(SEQ ID NO.: 2) into a reca positive cell strain resulted in geneticrecombination of the mutant MetRS gene with the chromosomal copy of thewild-type MetRS gene, yielding plasmid pQE 15-MRS (SEQ ID NO.: 1).

[0188] Expression plasmid pQEl5-MRS (SEQ ID NO.: 1) and repressorplasmid pREP4 were transformed into the expression host B834(DE3) toyield B834(DE3)/pQE15-MRS/pREP4. Plasmid DNA from allB834(DE3)/pQE15-MRS/pREP4 cultures used for protein expressionexperiments was sequenced to confirm that it encoded wild-type MetRS.The expression plasmid pQE15-MRS (SEQ ID NO.: 1) encodes MetRS undercontrol of the E. coli promoter metGpl (Genbank accession number X55791)(F. Dardel, M. Panvert, G. Fayat, Mol. Gen. Genet. 1990, 223, 121-133)as well as the target protein murine dihydrofolate reductase (mDHFR)under control of a bacteriophage T5 promoter. The expression plasmidalso encodes an N-terminal hexahistidine sequence for mDHFR whichpermits purification of the target protein by immobilized metal chelateaffinity chromatography (The Qiagen Expressionist, 1992, p. 45).Furthermore, mDHFR contains 8 methionine residues which can be replacedby methionine analogues. A control bacterial host, which produces onlymDHFR and normal cellular levels of MetRS, was prepared by transformingB834(DE3) with pREP4 and pQE1 5.

[0189] Similarly, a bacterial host capable of overexpressing MetRS wasproduced by transforming E. coli strains CAG18491 (Novagen, Inc.,Madison, Wis., USA), a methionine auxotroph, with repressor plasmidpREP4 and expression plasmid pQE15-MRS, as described for the B834(DE3)strain. A control bacterial host, which produces only mDHFR and normalcellular levels of MetRS, was prepared by transforming CAG18491withpREP4 and pQE15.

[0190] Methionine analogues 2-13 were tested for translational activityin both bacterial hosts. Methionine analogues were synthesized viaalkylation of diethylacetamidomalonate, as previously described (Asdescribed in Example I, infra; J. C. M. van Hest, D. A. Tirrell, FEBSLett. 1998, 428, 68-70). Cultures of B834(DE3)/pQEI5-MRS/pREP4 orB834(DE3)/pQE 15/pREP4 or CAG18491/pQE15-MRS/pREP4 orCAG18491/pQE15/pREP4 in M9AA media were grown to an optical density of0.90, and the cells were sedimented by centrifugation. The M9AA mediumwas prepared by supplementing sterile M9 medium with 60 mg/ml of each ofthe amino acids, 1 mM MgSO₄, 0.2 wt % glucose, 1 mg/ml thiaminechloride, and 1 mg/ml calcium chloride. The antibiotics ampicillin andkanamycin were added at concentrations of 200 mg/l and 35 mg/l,respectively.

[0191] Cells were washed three times with M9 salts and resuspended to anoptical density of 0.90 in M9 test media containing 19 amino acidsplus 1) neither methionine nor analogue (negative control); 2)methionine (60 mg/liter, positive control); or 3) an analogue ofinterest (60 mg/liter). To test the effect of increasing the level ofsupplementation of the analogues, a set of experiments was alsoconducted in which the medium was supplemented with 500 mg/liter ofmethionine or the amino acid analogue. Expression of mDHFR was inducedby addition of 0.4 mM isopropyl β-D-thiogalactopyranoside (IPTG), andprotein synthesis was monitored after 4.5 hours. Expression of mDHFR wasmonitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE);accumulation of target protein was taken as evidence for translationalactivity of the methionine analogue.

[0192] For cultures supplemented with amino acids at 60 mg/liter, thetarget protein was not observed in the negative control culture ofB834(DE3)/pQE15/pREP4, CAG18491/pQE15/pREP4 or in cultures supplementedwith Ccg (4), 6,6,6-trifluoro-2-aminohexanoic acid (6), 2-aminoheptanoicacid (7), norvaline (8) o-allylserine (10), allylgylcine (12) orpropargylglycine (13). In contrast, mDHFR was detected in both bacterialhost cultures supplemented with methionine (1), Hag (2), Hpg (3), andnorleucine (9), as indicated by the appearance of a protein band at theposition expected for mDHFR in SDS-PAGE.

[0193] For the negative control cultures and for cultures supplementedwith Tcg however, the behavior of the bacterial hosts differed, as shownin FIG. 7. mDHFR was not detected in the B834(DE3)/pQE15/pREP4 culturesupplemented with Tcg, while strong induction of mDHFR was observed forB834(DE3)/pQE15-MRS/pREP4 under the same conditions. Even theunsupplemented control culture of B834(DE3)/pQE15-MRS/pREP4 showsevidence of mDHFR synthesis, suggesting that introduction of pQE15-MRS(SEQ ID NO.: 1) does indeed increase the rate of activation ofmethionine in the modified host.

[0194] B834(DE3)/pQE15-MRS/pREP4 cells, which overexpress MetRS, havesufficient MetRS activity to synthesize measurable levels of proteinfrom the very low intracellular levels of methionine in the negativecontrol culture. Interestingly, aminoacyl-tRNA synthetase overexpressionis induced by amino acid starvation in some gram-positive bacteria,presumably to permit continued protein synthesis (D. Luo, J. Leautey, M.Grunberg-Manago, H. Putzer, J. Bacteriol. 1997, 179, 2472-2478).B834(DE3)/pQE15/pREP4 cultures, which lack the increased MetRS activity,do not show background expression of protein in negative controlcultures.

[0195] Similar results were observed for the CAG18491/pQE15/pREP4 andCAG18491/pQE15-MRS/pREP4 cultures supplemented with 11 (FIG. 8). WhilemDHFR was not detected in the CAG18491/pQE15/pREP4 cultures supplementedwith 11, strong induction of mDHFR was observed forCAG18491/pQE15-MRS/pREP4 under the same conditions. The unsupplementedcontrol culture of CAG18491/pQEI5-MRS/pREP4 showed little evidence ofmDHFR synthesis, which may be due to lower levels of MetRS activity inthese cell strains versus that in the B834(DE3)/pQE15-MRS/pREP4.

[0196] For cultures supplemented with amino acids at 500 mg/liter,however, the target protein mDHFR could be observed for certain aminoacid analogues only in cultures of cellular hosts containing the MetRS(FIG. 9). FIG. 9 demonstrates that the modified bacterial hostsCAG18491/pQE15-MRS/pREP4 are able to produce the target protein incultures supplemented with 500 mg/liter of 4, 7, 8, and 12. Quantitativecharacterization of the kinetic parameters of these analoguesdemonstrates that although the analogues do not support measurablelevels of PP₁ exchange after 20 minutes, they are activated by the MetRSin vitro (FIG. 10). Due to the very slow rate of activation supported bythese analogues, increasing the concentration of the analogues in themedium must be combined with introduction of pQE15-MRS (SEQ ID NO.: 1)into the bacterial host in order to raise the rate of activation ofthese analogues sufficiently to permit protein biosynthesis.

[0197] To confirm the supposition that the MetRS activity of a cellularhost is increased by the introduction of pQE15-MRS, direct measurementof the MetRS activities of whole cell lysates was conducted.B834(DE3)/pQEI5-MRS/pREP4 exhibits a V_(max) for methionine activationapproximately 30-fold higher than that observed for the control hostB834(DE3)/pQE15/pREP4 (FIG. 11). Similarly, CAG18491/pQE15-MRS/pREP4exhibits a V_(max) for methionine activation approximately 50-foldhigher than that observed for the control host CAG18491/pQE15/pREP4.ATP-PP_(i) exchange assays were conducted using the methods as describedsupra. A 50-μl aliquot of whole cell lysate with a normalized OD₆₀₀ of20 was prepared by one freeze-thaw cycle and added to the assay mixtureto yield a final volume of 150 μl. A saturating concentration ofmethionine (750 μM) was used to determine the maximum exchange velocityfor each cell lysate.

[0198] These results show clearly that increasing the MetRS activity ofthe host is necessary and sufficient to observe translational activityof 4, 5, 7, 8, 11, and 12 under convenient conditions in vivo. Proteinyields (mDHFR-Tcg) of approximately 8.5 mg/liter were observed forB834(DE3)/pQEI5-MRS/pREP4 cultures supplemented with Tcg, compared withyields of approximately 35 mg/liter for both B834(DE3)/pQEI5-MRS/pREP 4and B834(DE3)/pQEI5/pREP4 cultures supplemented with methionine. Aminoacid analysis of protein containing Tcg shows a decrease in methioninecontent to 0.3 mol % from the expected value of 3.8 mol %. It was notpossible to detect Tcg directly by amino acid analysis, owing toinstability of the analogue under the analysis conditions. If depletionof methionine is assumed to result from replacement by Tcg, the observedanalysis corresponds to an overall extent of incorporation of theanalogue of 91±2%. Amino acid analysis of mDHFR containing the otheranalogues (4, 7, 8, 11, and 12), showed 92-98% replacement ofmethionine.

[0199] A direct assessment of the extent of incorporation of Tcg intomDHFR was provided by NMR spectroscopy. Proton NMR spectra were recordedusing a Varian Inova NMR spectrometer with proton acquisition at 599.69MHz. Spectra were recorded at 25° C. overnight. A simple presaturationpulse was used for water suppression. Comparisons of the 600 MHz protonNMR spectra (FIG. 12) of mDHFR, Tcg, and mDHFR-Tcg indicate theappearance, in the mDHFR-Tcg spectrum (FIG. 12c), of the Tcg vinyleneprotons at δ=5.35(δ-CH) and δ=5.60-5.70 (γ-CH). The resonances at δ=5.35and δ=5.70 occur at the same chemical shift values as in free Tcg andare clearly due to incorporation of Tcg into mDHFR. That the resonanceat δ=5.60 arises from the γ-CH vinylene proton of Tcg is suggested bythe fact that the integrated intensity of the resonance at δ=5.35 equalsthe sum of the integrations of the resonances at δ=5.60 and δ=5.70. Thisassignment is confirmed by ID TOCSY (Total Correlation Spectroscopy)experiments which indicate that the protons at both δ=5.60 and δ=5.70are members of the same spin system (and therefore the same amino acid)as those at δ=5.35. More importantly, the ID TOCSY experiments also showthat the protons at δ=5.35 (and therefore those at δ=5.60 and δ=5.70)are associated with the spin system of the entire Tcg side chain (1DTOCSY spectra were recorded on a Varian Inova NMR spectrometer withproton acquisition at 599.69 MHz).

[0200] A 1D TOCSY pulse sequence (D. Uhrin, P. N. Barlow, J. Magn.Reson. 1997, 126, 248-255) with selective irradiation of the resonanceat δ=5.35 (E. Kupce, J. Boyd, I. D. Campbell, J. Magn. Reson. Ser. B.1995, 106, 300-303) was used to identify which protons belonged to thespin system of the δ=5. 35 resonance. The selectivity of the pulse isdemonstrated in a separate, simple 1D experiment in which the selectivepulse was applied alone; no other resonances were observed in thespectrum under these conditions. Observation after a mixing time of 60ms, however, showed the protons at δ=5.60 and δ=5.70, indicating thatthose protons are members of the same spin system (and therefore thesame amino acid residue) as those corresponding to the resonance atδ=5.35. The α-carbon and side chain β-and ε-carbon protons were alsoobserved at chemical shift values characteristic of the free amino acid(δ=4.3 (α-CH), 2.5 (β-CH2), and 1.6 (δ-CH3)). Integration of thespectrum suggests that 5 of the 8 methionine positions (occupied by Tcg)are represented by the resonance at δ=5.60; these protons must reside ina magnetically-distinct environment from the protons at (δ=5.70. Theseresults unequivocally demonstrate the translational activity of Tcg inthe host strain outfitted with elevated MetRS activity. Integration ofthe NMR spectrum indicates 90±6% replacement of methionine.

[0201] Retention of the N-terminal (initiator) methionine in mDHFR wasexpected on the basis of the identity of the penultimate amino acid (P.H. Hirel, J. M. Schmitter, P. Dessen, G. Fayat, S. Blanquet, Proc. Natl.Acad. Sci. USA 1989, 86, 8247-8251), so N-terminal sequencing provided athird means of assessing the extent of replacement of methionine byanalogues 4, 5, 7, 8, 11, and 12. Because the analogues were notdegraded under the analysis conditions, they could be detected directly.Comparison of chromatograms of the N-terminal residues of mDHFR andmDHFR-Tcg (FIG. 13) demonstrate that the methionine that normallyoccupies the initiator position of mDHFR (FIG. 13a) was nearlycompletely replaced with Tcg (FIG. 13b) in mDHFR-Tcg (FIG. 13c). Thesignal corresponding to methionine eluted at 13.8 min while thatcorresponding to Tcg eluted at 16.0 min. The large peaks (pptu) whichelute at approximately 15.4 min correspond to piperidylphenylthiourea, aproduct of the analysis resulting from the buffer, and the small peak(diet) at 19.4 min corresponds to diethylphthalate, an internalstandard. These results clearly indicate the incorporation of Tcg at theinitiator site of mDHFR-Tcg and corroborate the NMR results. Integrationof the peak areas corresponding to Tcg and to methionine indicates 96±2%incorporation of the analogue at the initiator position. Similaranalysis of mDHFR-2bg (2-butynylglycine) by N-terminal sequencingindicates 98±2% replacement of methionine (FIG. 4). Analysis of mDHFRcontainining analogues 8 and 12 shows replacement of methionine by theseanalogues at levels of 85-90%.

[0202] The incorporation of 4, 5, 7, 8, 11, and 12 into proteins in vivoconstitutes the first example of broadening the amino acid substraterange of the E. Coli translational apparatus via overproduction of MetRSin a bacterial host. The utilization of 4, 5, 7, 8, 11, and 12 in allstages of protein synthesis (including initiation) indicates theappropriateness of targeting the aminoacyl-tRNA synthetase in studiesaimed at in vivo incorporation of amino acid analogues into proteins.Transport into the cell, recognition by methionyl-tRNA formylase, andrecognition by the elongation factors and the ribosome are less likelyto be limiting factors.

[0203] These results indicate that this simple strategy ofoverexpression of aminoacyl-tRNA synthetase may be used to modifyproteins by incorporation of amino acid analogues that are poorsubstrates for aminoacyl-tRNA synthetase and that would be essentiallyinactive in conventional expression hosts.

[0204] Overexpression of mutant forms of the aminoacyl-tRNA synthetaseprepared via site-directed mutagenesis or directed evolution (F. H.Arnold, J. C. Moore, Adv. Biochem. Eng. Biotech. 1997, 58, 1-14; F. H.Arnold, Chem. Eng. Sci. 1996, 51, 5091-5102) should provide additionalstrategies for incorporating amino acid analogues into proteins in vivo.

[0205] The results reported here also suggest new opportunities formacromolecular synthesis via protein engineering. The versatilechemistry of unsaturated functional groups (B. M. Trost, I. Fleming,,Comprehensive Organic Synthesis, Pergamom Press, Oxford, 1991) can beused to control protein structure and function through chemicalderivatization, an especially intriguing possibility in this case giventhe important role of methionine in protein-protein recognitionprocesses. For example, ruthenium-catalyzed olefin metathesis (D. M.Lynn, B. Mohr, R. H. Grubbs, J. Am. Chem. Soc. 1998, 120, 1027-1028; E.L. Dias, T. N. SonBinh, R. H. Grubbs, J. Am. Chem. Soc. 1997, 119,3887-3897) of homoallylglycine (T. D. Clark, M. R. Ghadiri, J. Am. Chem.Soc. 1995, 117, 12364-12365) and o-allylserine (H. E. Blackwell, R. H.Grubbs, Angew. Chem. 1998, 110, 3469-3472; Angew. Chem. Int. Ed. 1998,37, 3281-3284) side chains has been used to produce covalently-modifiedpeptide structures of various kinds. The incorporation of Tcg may besingularly useful in this regard as the internal olefin is active inaqueous-phase ring closing metathesis reactions, whereasterminally-unsaturated groups (such as those previously used to replacemethionine in vivo) are not (T. A. Kirkland, D. M. Lynn, R. H. Grubbs,J. Org. Chem. 1998, 63, 9904-9909).

Example III

[0206] This example demonstrates that activation of methionine analoguesin vitro correlates well with the ability of these analogues to supportprotein synthesis in vivo, substantiating the critical role ofaminoacyl-tRNA synthetase in controlling the incorporation of amino acidanalogues into proteins.

[0207] Reagents

[0208] Each of the analogues 2-7 and 11 (FIG. 2) was prepared byalkylation of diethyl acetamidomalonate with the appropriate tosylatefollowed by decarboxylation and deprotection of the amine function (asdescribed in Example I). Methionine and analogues 8, 9, 12 and 13 wereobtained from Sigma (St. Louis, Mo). Radiolabeled sodium pyrophosphatewas purchased from NEN Life Science Products, Inc., andisopropyl-β-D-thiogalactopyranoside was obtained from Calbiochem. TheRGS-His antibody and anti-mouse IgG horseradish peroxidase conjugateused for Western blotting procedures were obtained from Qiagen andAmersham Life Sciences, respectively. All other reagents used duringprotein biosynthesis and purification and for activation assays werecommercially available from Sigma, Aldrich, and Qiagen, and were used asreceived.

[0209] In Vitro Activation Assays

[0210] The fully active, truncated form of MetRS was purified fromovernight cultures of E. coli JM101 cells carrying the plasmid pGG3(Kim, H. Y.; Ghosh, G.; Schulman, L. H.; Brunie, S.; Jakubowski, H.Proc. Natl. Acad. Sci. USA 1993, 90, 11553-11557), by using sizeexclusion methods previously reported (Mellot, P.; Mechulam, Y.;LeCorre, D.; Blanquet, S.; Fayat, G. J. Mol. Biol. 1989, 208, 429-443).Purified enzyme solutions (in 10 mM phosphate, pH 6.7, 10 mMβ-mercaptoethanol) were concentrated to at least 3 μM prior to theirstorage in 40% glycerol at −20° C. Concentrations of enzyme stocks weredetermined by the Bradford method, using samples of MetRS quantified byamino acid analysis as standards.

[0211] Activation of methionine analogues by MetRS was assayed via theamino acid-dependent ATP-PP_(i) exchange reaction at room temperature,also as previously described (Mellot, P.; Mechulam, Y.; LeCorre, D.;Blanquet, S.; Fayat, G. J. Mol. Biol. 1989, 208, 429-443; Ghosh, G.;Pelka, H.; Schulman, L. D. Biochemistry 1990, 29, 2220-2225). The assay,which measures the ³²P-radiolabeled ATP formed by the enzyme-catalyzedexchange of ³²P-pyrophosphate (PP_(i)) into ATP, was conducted in 150 tlof reaction buffer (pH 7.6, 20 mM imidazole, 0.1 mM EDTA, 10 mMβ-mercaptoethanol, 7 mM MgCI₂, 2 mM ATP, 0.1 mg/ml BSA, and 2 mM PP_(i)(in the form of sodium pyrophosphate with a specific activity ofapproximately 0.5 TBq/mole)).

[0212] Kinetic parameters for methionine analogues 2, 3, 5, 9 and 11were obtained with an enzyme concentration of 75 nM and analogueconcentrations of 100 μM to 20 mM. Parameters for methionine wereobtained by using methionine concentrations ranging from 10 μM to 1 mM.Aliquots (20 μl) were removed from the reaction mixture at various timepoints and quenched in 0.5 ml of a solution comprising 200 mM PP_(i), 7%w/v HClO₄, and 3% w/v activated charcoal. The charcoal was rinsed twicewith 0.5 mL of a 10 mM PP_(i), 0.5% HClO₄ solution and then resuspendedin 0.5 mL of this solution and counted via liquid scintillation methods.Kinetic constants were calculated by a nonlinear regression fit of thedata to the Michaelis-Menten model.

[0213] In Vivo Incorporation of Amino Acid Analogues

[0214] Buffers and media were prepared according to standard protocols(Ausubel, F. M.; Brent, R.; Kingston, R. E.; Moore, D. D.; Seidman, J.G.; Smith, J. A.; Struhl, K., Eds. Current Protocols in MolecularBiology; John Wiley and Sons: New York, 1998). The E. coli methionineauxotroph CAG18491 (λ, rph-1, metE3079.:Tn10) was transformed withplasmids pQE15 and pREP4 (Qiagen), to obtain the expression hostCAG18491/pQE15/pREP4. The auxotroph was transformed with the plasmidspQE15-MRS (SEQ ID NO.: 1) and pREP4 to obtain the modified bacterialexpression host CAG18491/pQE15-MRS/pREP4. Both bacterial expressionhosts produce the target protein mDHFR under control of a bacteriophageT5 promoter; the modified host also expresses extra copies of the MetRSgene under control of the constitutive metG p1 promoter (Dardel, F.;Panvert, M.; Fayat, G. Mol. Gen. Genet. 1990, 223, 121-133).

[0215] Protein Expression (1 Liter Scale).

[0216] Similar procedures were used for preparation and isolation ofmDHFR from media supplemented with the L-isomers of 1, 2, 3, or 9. M9AAmedium (100 mL) supplemented with 1 mM MgSO₄ 0.2 w % glucose, 1 mg/Lthiamine chloride and the antibiotics ampicillin (200 mg/L) andkanamycin (35 mg/L) was inoculated with the appropriate E. coli strain(CAG18491/pQE15/pREP4 or CAG18491/pQE15-MRS/pREP4) and grown overnightat 37° C. This culture was used to inoculate 900 mL M9AA mediumsupplemented as described. The cells were grown to an optical density at600 nm (OD₆₀₀) of approximately 0.9 and a medium shift was performed.The cells were sedimented for 10 min at 3030 g at 4° C., the supernatantwas removed, and the cell pellet was washed twice with 600 mL of M9medium. Cells were resuspended in 1000 mL of the M9AA medium describedabove, without methionine, and supplemented with 20 mg/L of the L-isomerof either 1, 2, 3, or 9. Protein synthesis was induced by addition ofisopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of0.4 mM. Samples (1 mL) were collected after 4.5 hours, the OD₆₀₀measured, and cells resuspended with distilled water to yield anormalized OD₆₀₀ of 20. Protein expression was monitored by SDSpolyacrylamide gel electrophoresis (12% acrylamide running gel);accumulation of mDHFR could be observed at an apparent molar mass ofapproximately 28 kDa after Coomassie staining.

[0217] Protein Purification.

[0218] Approximately 4.5 h after induction, cells were sedimented (9,800g, 10 min, 4° C.) and the supernatant was removed. The pellet was placedin the freezer overnight. The cells were thawed for 30 min at 37° C., 30mL of buffer (6 M guanidine-HCl, 0.1 M NaH₂PO₄, 0.01 M Tris, pH 8) wasadded and the mixture was shaken at room temperature for 1 h. The celldebris was sedimented (15,300 g, 20 min, 4° C.) and the supernatant wassubjected to immobilized metal affinity chromatography (Ni-NTA resin)according to the procedure described by Qiagen (The QiagenExpressionist; Qiagen; Valencia, Calif., 2000). The supernatant wasloaded on 10 mL of resin which was then washed with 50 mL of guanidinebuffer followed by 25 mL of urea buffer (8 M urea, 0.1 M NaH₂PO₄ and0.01 M Tris, pH 8). Similar urea buffers were used for three successive25 mL washes at pH values of 6.3, 5.9 and 4.5, respectively. Targetprotein was obtained in washes at pH 5.9 and 4.5. These washes werecombined and dialyzed (Spectra/Por membrane 1, MWCO=6-8 kDa) bybatchwise dialysis against doubly distilled water for 4 days with atleast 12 total changes of water. The dialysate was lyophilized to apurified powder of mDHFR. Experiments in M9AA medium affordapproximately 30 mg of mDHFR for each of the bacterial expression hosts,while a control experiment in 2×xYT medium afforded approximately 60 mgof mDHFR. Protein yields are reported as mg protein obtained per literof bacterial culture; approximately 5-6 g of wet cells are obtained perliter of culture regardless of the identity of the analogue used tosupplement the medium.

[0219] Protein Expression (5 mL Scale).

[0220] M9AA medium (50 mL) supplemented with 1 mM MgSO₄, 0.2 wt %glucose, 1 mg/L thiamine chloride and the antibiotics ampicillin (200mg/L) and kanamycin (35 mg/L) was inoculated with 5 mL of an overnightculture of the appropriate bacterial expression host. When the turbidityof the culture reached an OD₆₀₀ of 0.8, a medium shift was performed.The cells were sedimented for 10 min at 3030 g at 4° C., the supernatantwas removed, and the cell pellet was washed twice with 25 mL of M9medium. Cells were resuspended in 50 mL of the M9AA medium describedabove, without methionine. Test tubes containing 5 mL aliquots of theresulting culture were prepared, and were supplemented with 10 μL of 10mg/mL L-methionine (1) (positive control), L-homoallylglycine (2),L-homopropargylglycine (3), or L-norleucine (9), respectively. A culturelacking methionine (or any analogue) served as the negative control.Protein expression was induced by addition of IPTG to a finalconcentration of 0.4 mM. After 4 h, the OD₆₀₀ was measured, and thesamples were sedimented. After the supernatant was decanted, the cellpellets were resuspended in distilled water to yield a normalized OD of20.

[0221] Protein expression was monitored by SDS polyacrylamide gelelectrophoresis (12% acrylamide running gel), followed by Westernblotting. After transfer to a nitrocellulose membrane, Western blotswere developed by treatment with a primary RGS-His antibody, followed bytreatment with a secondary anti-mouse IgG conjugated to horseradishperoxidase to provide detection by chemiluminescence. Films were checkedto ensure that band intensity was not saturated. Levels of proteinsynthesis were estimated by the intensity of the band on the gel, asdetermined using a Pharmacia Ultrascan XL laser densitometer andanalysis by Pharmacia GelScan XL software. The accumulation of targetprotein is taken as evidence for incorporation of the amino acidanalogue, as 2, 3, 5, 9 and 11 have been shown to replace methionine,even in modified bacterial hosts, at levels of 92-98% (van Hest, J. C.M.; Tirrell, D. A. FEBS Lett. 1998, 428, 68-70; as described in ExampleI, infra; Budisa, N.; Steipe, B.; Demange, P.; Eckerskorn, C.;Kellermann, J.; Huber, R. Eur. J. Biochem. 1995, 230, 788-796).

[0222] Results and Discussion

[0223] Studies with methionine analogues 2-13 (as described in ExampleI, infra), demonstrated that 2 and 3 can be incorporated into proteinswith extents of substitution up to 98%. The incorporation of 9 had beenpreviously reported (Budisa, N.; Steipe, B.; Demange, P.; Eckerskorn,C.; Kellermann, J.; Huber, R. Eur. J. Biochem. 1995, 230, 788-796). Incontrast, 4-8 and 10-13 do not support protein synthesis in the absenceof methionine in a conventional bacterial expression host; investigationof the activation of the analogues by methionyl-tRNA synthetase (MetRS)indicated that 4-8 and 10-13 are not efficiently activated by theenzyme.

[0224] Overproduction of MetRS in the bacterial host, however, permitsincorporation of 4, 5, 7, 8, 11 and 12, which show very slow exchange ofPPi in in vitro activation assays (Example II, infra). These resultsindicate that the aminoacyl-tRNA synthetase are appropriate targets forstudies aimed at the incorporation of amino acid analogues into proteinsin vivo. The results also suggest that neither transport into the cellnor recognition by the elongation factors or the ribosome limits theincorporation of these amino acid analogues into proteins in vivo.

[0225] In this example, the in vitro activation of 2-13 by MetRS wascharacterized in order to determine the roles of the synthetase incontrolling analogue incorporation and protein yield in mediasupplemented with amino acid analogues. Furthermore, the analogues 2 and3, which replace methionine in vivo, may be useful for chemicalmodification of proteins by olefin metathesis (Clark, T. D.; Kobayashi,K.; Ghadiri, M. R. Chem. Eur. J. 1999, 5, 782-792; Blackwell, H. E.;Grubbs, R. H. Angew. Chem. Int. Ed. Engl. 1998, 37, 3281-3284),palladium-catalyzed coupling (Amatore, C.; Jutand, A. J. Organomet.Chem. 1999, 576, 255-277; Tsuji, J. Palladium Reagents and Catalysts:Innovations in Organic Synthesis; John Wiley and Sons: New York, 1995;Schoenberg, A.; Heck, R. F. J. Org. Chem. 1974, 39, 3327-3331), andother chemistries (Trost, B. M.; Fleming, I., Eds. Comprehensive OrganicSynthesis; Pergamon Press: Oxford, 1991).

[0226] The attachment of an amino acid to its cognate tRNA proceeds intwo steps (FIG. 18). Activation, the first step, involves theenzyme-catalyzed formation of an aminoacyl adenylate (designated aa˜AMPin FIG. 18) and can be examined by monitoring the rate of exchange ofradiolabeled pyrophosphate (³²P PP₁) into ATP (Fersht, A. Structure andMechanism in Protein Science; W. H. Freeman and Company: New York,1999). Aminoacylation, the second step, can be evaluated by monitoringthe amount of radiolabeled amino acid attached to tRNA in the presenceof the enzyme. Because initial recognition of an amino acid by itsaminoacyl-tRNA synthetase is perhaps the most critical step in theincorporation of amino acid analogues into proteins in vivo. Thus, thefocus has been on the in vitro activation of methionine analogues byMetRS was evaluated and the results compared to those obtained instudies of in vivo incorporation.

[0227] The rates of activation of 2-13 by MetRS were determined by theATP-PP_(i) exchange assay, and were found to correlate well with theresults of the in vivo studies; analogues 2-5, 7-9, 11 and 12 (thosewhich had been shown to support protein synthesis) exhibited measurableexchange of PP_(i) (Example I and II, infra). The kinetic parametersk_(cat) and K_(m) were determined for each of these analogues; theresults for analogues 2, 3, 5, 9 and 11 are summarized in FIG. 15. Themeasured K_(m) for methionine matched previously reported values (Kim,H. Y.; Ghosh, G.; Schulman, L. H.; Brunie, S.; Jakubowski, H. Proc.Natl. Acad. Sci. USA 1993, 90, 11553-11557). The value determined fork_(cat) was slightly lower than the literature value. Comparison of thek_(cat)/K_(m) values for each of the analogues with that for methionineshowed that these analogues were 500-fold to 13825-fold poorersubstrates for MetRS than methionine.

[0228]FIG. 15 also demonstrates that methionine analogues that areactivated up to 2000-fold more slowly by MetRS than methionine cansupport protein synthesis in a conventional bacterial host in theabsence of methionine. (Poorer substrates, such as 5, requiremodification of the MetRS activity of the bacterial host in order tosupport protein synthesis (Example II, infra). These results arecomparable to those reported previously for the activation and in vivoincorporation of phenylalanine analogues (Gabius, H. J.; von der Haar,F.; Cramer, F. Biochemistry 1983, 22, 2331-2339; Kothakota, S.; Mason,T. L.; Tirrell, D. A.; Fournier, M. J. J. Am. Chem. Soc. 1995, 117,536-537; Ibba, M.; Kast, P.; Hennecke, H. Biochemistry 1994, 33,7107-7112). Comparisons for other amino acids have been limited by alack of in vitro activation data. The data suggested that amino acidanalogues can support protein synthesis in vivo even with surprisinglyinefficient activation of the amino acid by its aminoacyl-tRNAsynthetase. Activation of methionine analogues by MetRS governs theirability to support protein synthesis in vivo.

[0229] Based on these results, it seemed likely that the kinetics ofanalogue activation would limit the rate and yield of protein synthesisin bacterial cultures supplemented with methionine analogues that arepoor substrates for MetRS. This correlation was investigated bycomparing the kinetic constants for analogue activation by MetRS withthe yield of the target protein murine dihydrofolate reductase (mDHFR)obtained from 1 -liter cultures of the bacterial hostCAG18491/pQE15/pREP4.

[0230] The CAG18491/pQE15/pREP4 bacterial host was produced bytransforming the E. coil methionine auxotroph CAG18491 with theexpression plasmid pQE15 and the repressor plasmid pREP4. The expressionplasmid pQE15 encodes mDHFR under control of a bacteriophage T5 promoterand an N-terminal hexahistidine sequence that permits purification ofthe target protein by immobilized metal chelate affinity chromatography(The Qiagen Expressionist; Qiagen; Valencia, Calif., 2000).

[0231] The kinetic constants for analogue activation and thecorresponding protein yields are listed in FIG. 15 and shown in FIG. 16.Analogues with the highest k_(cat)/K_(m) values also support the highestlevels of protein synthesis; the protein yields scale remarkably wellwith k_(cat)/K_(m), at least for the poorer substrates. Analogue 3supported protein synthesis with yields equivalent to those obtainedwith methionine, despite the fact that 3 is a 500-fold poorer substratefor MetRS than methionine.

[0232] Bacterial cultures supplemented with 9 (1050-fold lowerk_(cat)/K_(m)) produce 57% as much mDHFR as cultures supplemented withmethionine, and cultures supplemented with 2 (1850-fold lowerk_(cat)/K_(m)) produce 28% of the control yield of protein. Bacterialcultures supplemented with 5 (4700-fold lower k_(cat)/K_(m)) and 11(13825-fold lower k_(cat)/K_(m)) did not support measurable levels ofprotein synthesis in this expression host; however, bacterial hostsexhibiting approximately 30-fold higher MetRS activity produce 23% asmuch mDHFR in cultures supplemented with 5 as cultures supplemented withmethionine (Example II, infra).

[0233] These results demonstrate that the rate of methionine analogueactivation in vitro does indeed correlate with protein yield in vivo.The results suggest that the kinetics of activation can play a criticalrole in controlling the rate of protein synthesis in methionine-depletedcultures supplemented with analogues that are poor substrates for MetRS.

[0234] Protein yields obtained from bacterial cultures supplemented withmethionine analogues might be improved by increasing the MetRS activityof the bacterial host. To test this, the yields of protein prepared werecompared in the conventional bacterial expression host,CAG18491/pQE15/pREP4, to those obtained from a modified host,CAG18491/pQE1 5-MRS/pREP4.

[0235] The modified CAG18491/pQE15-MRS/pREP4 host was prepared bytransforming E. coli strain CAG18491 with the expression plasmidpQE15-MRS (SEQ ID NO.: 1) (Example II, infra) and the repressor plasmidpREP4. The expression plasmid pQE15-MRS (SEQ ID NO.: 1) encodes MetRSunder control of the E. coli promoter metG p1 (Genbank accession numberX55791) (Dardel, F.; Panvert, M.; Fayat, G. Mol. Gen. Genet. 1990, 223,121-133) as well as the target protein mDHFR. The MetRS activity of thebacterial hosts was determined as previously described (Example II,infra), with the modified host exhibiting 50-fold higher MetRS activitythan the conventional strain.

[0236] Protein synthesis was monitored for 5-ml cultures of these hostssupplemented with methionine or analogues 2, 3, or 9. Western blotanalyses of protein synthesis are shown in FIG. 17. Although very lowlevels of protein synthesis were observed for negative control culturesof CAG18491/pQE15-MRS/pREP4, amino acid analyses, N-terminal sequencing,and NMR analyses of proteins produced in cultures of the modified hostsupplemented with 5 and 11 (the poorest of the substrates) still showed90-96% replacement of methionine by 5 and 11 (Example II, infra). Thus,the level of protein synthesis shown in FIG. 17 resulted from theincorporation of the analogue and was not due to incorporation ofresidual methionine.

[0237] For cultures supplemented with methionine or 3, the modifiedhost, CAG18491/pQEI15-MRS/pREP4, does not exhibit higher levels ofprotein synthesis than the conventional host CAG18491/pQE15/pREP4.Analysis by laser densitometry confirmed these results, and revealedapproximately equal accumulation of target protein for both strains;identical results have been obtained for large-scale expressions andpurification of mDHFR. Activation of the analogue by MetRS does notappear to limit protein synthesis in cultures supplemented with 3. Forcultures supplemented with 2 or 9, however, the modified bacterial hostexhibits significantly increased levels of protein synthesis incomparison with the conventional host. Laser densitometry analysisindicates that the level of protein synthesis in the modified host isincreased approximately 1.5-fold over that in the conventional host forcultures supplemented with 2, and approximately 1.4-fold for culturessupplemented with 9. Activation of these analogues by MetRS appears tolimit protein synthesis in the conventional host, such that increasingthe MetRS activity of the host is sufficient to restore high levels ofprotein synthesis. Preliminary results indicated that the yield of mDHFRobtained from large-scale cultures of CAG18491/pQE15-MRS/pREP4supplemented with 2 or 9 are increased to approximately 35 mg/L (from 10mg/L obtained from cultures of CAG18491/pQE15/pREP4 (FIG. 15)).

[0238] The results indicate that overexpression of MetRS can improveprotein yields for cultures supplemented with methionine analogues thatare poor substrates for MetRS, and provide an attractive general methodfor efficient production of chemically novel protein materials in vivo.

[0239] Quantitative assessment of the kinetics of activation by MetRShave indicated that even very poor substrates for the synthetase can beutilized by the protein synthesis machinery of a bacterial expressionhost. The correlation, shown herein, between the in vitro and in vivoresults indicates the important role of the aminoacyl-tRNA synthetaseand suggests that site-directed mutatgenesis and/or directed evolutionof this class of enzymes may be used to increase further the number ofamino acid analogues that can be incorporated into proteins in vivo.

[0240] These results also indicate that the kinetics of activation ofmethionine analogues by MetRS in vitro correlate with the level ofprotein synthesis supported by the analogues in vivo. The activity ofthe MetRS in the bacterial host can be manipulated, by overexpression ofthe MetRS, to improve the yields of proteins containing methionineanalogues that are poor substrates for the MetRS. Overexpression ofaminoacyl-tRNA synthetase used to improve yields of proteins containingother amino acid analogues, as well as proteins rich in particularnatural amino acids. Manipulation of the aminoacyl-tRNA synthetaseactivities of a bacterial host broadens the scope of proteinengineering, by permitting production of natural and artificialproteins, with novel chemical and physical properties.

What is claimed:
 1. A method for producing a modified polypeptide, thepolypeptide being modified by replacing a selected amino acid with adesired amino acid analogue, which method comprises: a. transforming ahost cell with: i. a vector having a polynucleotide sequence encoding anaminoacyl-tRNA synthetase for the selected amino acid; and ii. a vectorhaving a polynucleotide sequence encoding a polypeptide molecule ofinterest so as to produce a host vector system; wherein the vectors of(i) and (ii) may be the same or different b. growing the host-vectorsystem in a medium which comprises the selected amino acid so that thehost vector system overexpresses the aminoacyl-tRNA synthetase. c.replacing the medium with a medium which lacks the selected amino acidand has the desired amino acid analogue. d. growing the host vectorsystem in the medium which lacks the selected amino acid and has thedesired amino acid analogue under conditions so that the host vectorsystem overexpresses the polypeptide molecule of interest and theselected amino acid is replaced with the desired amino acid analoguethereby producing the modified polypeptide.
 2. The method of claim 1wherein the overexpression of an aminoacyl-tRNA synthetase results in anincrease in the activity of the aminoacyl-tRNA synthetase.
 3. The methodof claim 1 wherein said host cell is from an organism which is selectedfrom a group consisting of bacterial, yeast, mammalian, insect, orplant.
 4. The method of claim 1 wherein said host cell is an auxotroph,the auxotrophic host cell incapable of producing the selected aminoacid.
 5. The method of claim 4 wherein said auxotrophic host cell isfrom an organism which is selected from a group consisting of bacterial,yeast, mammalian, insect, or plant.
 6. The method of claim 5, whereinthe said auxotrophic host cell is a methionine auxotroph.
 7. The methodof claim 1, wherein said aminoacyl-tRNA synthetase is naturallyoccurring or genetically engineered.
 8. The method of claim 1, whereinsaid aminoacyl-tRNA synthetase is methionyl tRNA synthetase.
 9. Themethod of claim 1, wherein said selected amino acid is methionine. 10.The method of claim 1, wherein said desired amino acid analoguecomprises side chain functionalities different from its correspondingnatural amino acid.
 11. The method of claim 10, wherein said amino acidanalogue is a hydrophobic amino acid analogue.
 12. The method of claim11, wherein the hydrophobic amino acid analogue is selected from thegroup consisting of fluorinated, electroactive, and unsaturated aminoacids.
 13. The method of claim 1, wherein the polypeptide isdihydrofolate reductase protein.
 14. The method of claim 1, wherein theselected amino acid is methionine; and the desired amino acid analogueis selected from the group consisting of homoallyglycine,homoproparglycine, norvaline, norleucine, cis-crotylglycine,trans-crotylglycine, 2-aminoheptanoic acid, 2-butynylglycine,allylglycine, azidoalanine and azidohomoalanine.
 15. A polypeptidemolecule produced by the method of claim
 1. 16. A dihydrofolatereductase protein produced by the method of claim
 13. 17. A recombinantvector comprising a polynucleotide sequence encoding an aminoacyl-tRNAsynthetase for the selected amino acid and a polynucleotide sequenceencoding a polypeptide molecule of interest.
 18. The recombinant vectorof claim 17, wherein said polynucleotide sequence encoding anaminoacyl-tRNA synthetase for the selected amino acid encodes formethionyl tRNA synthetase.
 19. The recombinant vector of claim 17,wherein said a polynucleotide sequence encoding a polypeptide moleculeof interest encodes for dihydrofolate reductase protein.
 20. Anauxotroph host cell comprising the recombinant vector of claim
 17. 21.The auxotroph host cell of claim 20, wherein the auxotroph host cell isfrom an organism which is selected from the group consisting ofbacterial, yeast, mammalian, insect and plant cells.
 22. A host cellcomprising the recombinant vector of claim
 17. 23. The host cell ofclaim 22, wherein the host cell is from an organism which is selectedfrom the group consisting of bacterial, yeast, mammalian, insect andplant cells.